ISSN 0972-1045

Vol. 14, No. 1, January-June, 2012

Appl Hort

Journal of THE SOCIETY FOR ADVANCEMENT OF HORTICULTURE

JOURNAL OF APPLIED HORTICULTURE

Vol. 14, No. 1, January-June, 2012

CONTENTS
Increased regeneration ability of transgenic callus of carrot (Daucus carota L.) on B5-based regeneration medium Yuan-Yeu Yau and Kevin Yueju Wang (USA) Avoiding the use of plant growth regulator in geranium production by application of a cyclic decit irrigation strategy Patrick Riga (Spain) Effect of light emitting diodes (LEDs) on postharvest needle retention of balsam r (Abies balsamea L.) R. Scott Veitch, Rajasekaran R. Lada and Mason T. MacDonald (Canada) Effect of decit drip-irrigation scheduling regimes with saline water on pepper yield, water productivity and soil salinity under arid conditions of Tunisia K. Nagaz, M.M. Masmoudi and N. Ben Mechlia (Tunisia) Observations on leaf morphology of male and female Actinidia chinensis plants W. Liu, M. Yang and H. Liang (China) Growth and foliar nutrient concentration response of Clerodendrum thomsoniae to increasing fertilization Karen I. Davis, Carl E. Niedziela Jr., Brian E. Whipker and Muchha R. Reddy (USA) Inuences of severe water stress on photosynthesis, water use efciency and proline content of almond cultivars Kazem Barzegar, Abbas Yadollahi, Ali Imani and Noorollah Ahmadi (Iran) Selection of resistant source to early blight disease in tomato among the Solanum species A.K. Singh, N. Rai, R.K. Singh, Major Singh, R.P. Singh, Smita Singh and Satyandra Singh (India) Water requirement of pomegranate (Punica granatum L.) plants upto ve year age D.T. Meshram, S.D. Gorantiwar, H.K. Mittal, N.V. Singh and A.S. Lohkare (India) Effect of different mulch materials on the incidence and severity of okra mosaic virus (OMV) in okra K.T. Kareem, O.O. Alamu, R.K. Egberongbe and O. Arogundade (Nigeria) Effect of putrescine, GA3, 2, 4-D, and calcium on delaying peel senescence and extending harvest season of navel orange H.A. Kassem, H.A. Marzouk and R.S. Al-Obeed (Egypt) In vitro free radical scavenging activity of aonla (Emblica ofcinalis) varieties at various stages of fruit development S. Haripriya, E. Vadivel, R. Venkatachalam and P. Gayathri (India) Response of some Egyptian sweet melon (Cucumis melo var. Aegyptiacus L.) cultivars to water stress conditions E.A. Ibrahim (Egypt) Micropropagation of strawberry cultivar Sweet Charlie through axillary shoot proliferation R. Rekha, Pallavi Mandave and Neelambika Meti (India) Potential use of shea nut (Vitteleria paradoxom) butter as skin coat for ripening and improved storage of banana E.K. Tsado (Nigeria) Allelopathic effect of orchard soils on seedling growth of rough lemon (Citrus jambhiri Lush.) R.P.S. Dalal, Navjot, A. Thakur, A.S. Sidhu and J.S. Brar (India) 3

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Forthcoming Papers
Effect of irrigation levels on fruit quality of the Picual olive (Olea europaea L.) cultivar M.M. Khattab, A.E. Shaban, I. Hussein and O.H. Elgamaal (Egypt) Effect of root substrates and seed cover materials on the germination and growth of organic tomato transplants Kurt O. Taylora, Muchha R. Reddya, Carl E. Niedziela Jr., Mary M. Peetc and Godfrey Gaylea (USA) Overcoming the seasonality of production by growing two types of indeterminate tomato (Solanum lycopersicum) varieties under cooled plastic house conditions in SudanRanda B.M. Ali and Saifeldin Mohamed El-Amin (Sudan) Improvement of somatic embryogenesis and plant regeneration in date palm (Phoenix dactylifera L.): Effect of cytokinins and activated charcoal on seven date palm cultivarsMona M. Hassan, Ibrahim A. Ibrahim, Mohsen K.H. Ebrahim and Ewald Komor (Egypt) Isolation of biomolecules of pharmacological importance from Garcinia indica fruit and evaluation of total antioxidant activityP. Gayathri and P. Govindaraju (India) Seasonal changes in endogenous hormone and sugar contents during bud dormancy in tree peony Philip M.P. Mornya and Fangyun Cheng (China) Assessment of strength of self-incompatibility in the S-allele lines of cabbage (Brassica oleracea var. capitata L.) Saurabh Singh and Vidyasagar (India) Evaluation of the resistance of pistachio root-stocks to Meloidogyne species in Iran Mehrdad Madani, Ahmad Akhiani and Ahmad Kheiri (Iran) Arsenic accumulation in pumpkin through contaminated groundwater and varietal evaluation thereof in Gangetic alluvium of West Bengal Rajib Kundu, Sukanta Pal, Pintoo Bandhopadhyay and Aparajita Majumder (India) The role of avocado in coffee based farming system of south western Ethiopia: The case of Jimma Zone Berhnau Megerssa (Ethiopia) Enhancing water relations and vase life of cut tulip (Tulipa gesneriana L.) using oral preservatives R. Kumar, N. Ahmed, D.B. Singh and O.C. Sharma (India) Effect of different growth media on the growth and owering of Beef steak Begonia (Begonia erythrophylla) Henry A. Akintoye, Olusola O. AdeOluwa, Olukemi Y. Akinkunmi (Nigeria) Growth, yield and nutrient uptake responses of snake tomato (Trichosanthes cucumerina L.) to organo-mineral fertilizer rates and leaf harvest methods O.O. Olubode, R.I. Adedeji, and O.O. Oyegbola (Nigeria) Water retention characteristics of soil bio-amendments used as growing media in pot culture S S Kukal, Debasish-Saha, Arnab-Bhowmik and R K Dubey (India) Occurrence of false smut on date palm (Phaenix dacyilifera L.) in the southern coastal plain of Yemen M.H. Abdul Sattar, A. Rashid, Yassin Ibrahim and Watheq A. Aulaqi (Yemen) Postharvest microbial diversity on major cultivars of Indian mangoes S.N. Jha, Pranita Jaiswal, K. Narsaiah, Rishi Bhardwaj, Poonam Preet Kaur, Ashish Kumar Singh, Rajiv Sharma and R. Kumar (India) Resource use efciency of orange and kinnow cultivation in Jammu region of J&K state Jyoti Kachroo, Anil Bhat and Dileep Kachroo (India) Transformation of cabbage (Brassica oleracea var. capitata) expressing a synthetic cry1F gene resistant to diamondback moth (Plutella xylostella) Lepidoptera: Yponomeutidae H.M. Mahadeva Swamy, S.N. Nagesha, Riaz Mahmood, T.K.S. Gowda and R. Asokan (India) QTL analysis associated with oleoresin content in intraspecic RIL population of chilli (Capsicum annuum L.)Neeraj Dwivedi, Rajesh Kumar, Rakesh Kumar Singh and Major Singh (India)

Journal

Journal of Applied Horticulture, 14(1): 3-6, 2012

Appl

Increased regeneration ability of transgenic callus of carrot (Daucus carota L.) on B5-based regeneration medium
Yuan-Yeu Yau1,2* and Kevin Yueju Wang2
USDA-ARS Vegetable Research Crops Unit and Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706, USA. 2Present address: Department of Natural Resources, Northeastern State University, Broken Arrow, OK 74014, USA. *E-mail: yau@nsuok.edu
1

Abstract
The in vitro development of a whole plant from a single cell is a characteristic feature of plants. Successful embryogenesis and regeneration during in vitro tissue culture are inuenced by different factors including medium components. In this study, we compared two regeneration media (MSIII, B5) and a mixture of these media (MSIII+B5) for the regeneration of plants from putative transgenic carrot calli. Seventeen times more plantlets were regenerated on B5 medium than on either MSIII or MSIII+B5 medium. A total of 432 plantlets were regenerated on B5 medium, compared to only 24 and 28 plantlets on MSIII and MSIII+B5, respectively. Plantlets regenerated on B5 medium were generally healthier and bigger than those regenerated on either MSIII or MSIII+B5 medium. Fiftytwo plantlets, 7-9 cm in length, were observed on the B5 regeneration medium, while no plants having 7-9 cm length were observed on either MSIII or MSIII+B5 medium after 4 months. This study demonstrated that B5 is a better medium than MSIII or MSIII+B5 medium for carrot callus regeneration and can be used routinely and efciently for carrot genetic transformation experiments. The transgenic nature of the regenerated plants was conrmed by both GUS staining assay and Southern hybridization analysis. Key words: Agrobacterium, carrot, callus, genetic transformation, regeneration

Introduction
Carrot (Daucus carota L.) is one of the major vegetable crops produced around the world. According to the annual report from Food and Agriculture Organization (FAO) of the United Nations, approximately 20 million metric tons of carrots were produced worldwide in 2005, with China, Russia and the United States the top three producing countries (http://www.fao.org). Carrots represent a major source of vitamin A and fiber for human nutrition (Simon, 1997; Horvitz et al., 2004). The phytochemicals in carrots such as -carotene (provitamin A), lutein, lycopene and anthocyanins play an important nutritional role in human health (Seddon et al., 1994). In the past decades, traditional breeding methods have greatly contributed to the improvement of carrot traits such as root shape, root color, smooth skin, -carotene levels and sugar content (Ammirato, 1986; Simon et al., 1989; Yau and Simon, 2005). However, genetic transformation can be used as a complementary technology to improve carrot quality and productivity (Jayaraj et al., 2007). Although carrot is a model system for tissue culture studies on somatic embryogenesis, carrot is not considered as a model plant (like Arabidopsis and tobacco) for genetic transformation due to its prolonged periods of time for tissue-culturing and development of regenerated plants. The process of carrot transformation is lengthy and labour intensive. Current protocols for carrot transformation still have room for improvement. A more efcient genetic transformation system is desirable. Regeneration is an important part of plant tissue culture. A suitable regeneration medium to regenerate plants from callus is critical in performing tissue culture or genetic transformation (Shin et al., 2000; utar-Vozli et al., 1999; Shao et al., 2000).

For certain in vitro breeding programs, efcient regeneration is especially important to obtain a large number of healthy plantlets for growing or evaluation from calli which are cultured for a prolonged period (Albert et al., 1995; Winicov, 1996). Studies to improve plant regeneration from long-term cultured callus, such as in rice, including modifying medium have been reported (Yin et al., 1993; Yang et al., 1999). Although researchers have successfully used MS- (Murashige and Skoog, 1962) and B5- (Gamborg et al., 1976) based or modied regeneration media for callus induction and regeneration in carrot transformation to produce a small scale of transgenic carrots for the purpose of study under laboratory conditions (Wurtele and Bulka, 1989; Thomas et al., 1989; Gilbert et al., 1996; Hardegger and Sturm, 1998; Baranski et al., 2006), it will be useful to know which of these two regeneration media provide a better regeneration capacity when a large scale plantlet production is needed for commercial purpose. In addition, carrot is an outcross species with severe inbreeding depression and one way of maintaining a specic trait (especially those traits controlled by multiple genes) in the progeny is through tissue culture. The information of callus regeneration ability on existing media is important. A direct comparison of MS- and B5-based media for carrot callus regeneration has not been reported, even though B5 medium has been mentioned as the better medium for carrot callus induction (Hardegger and Sturm, 1998). The objective of this study was to evaluate the regeneration ability of transgenic callus tissues derived from carrot line B493 (Simon et al., 1990) on the media MSIII and B5, as well as on a mixture of MSIII and B5 (referred to as MSIII+B5). Carrot inbred line B493 was used for this experiment due to its ability in

Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium terminator-35S promoter-gus gene-Nos terminator- T-DNA left border was transferred into Agrobacterium tumefaciens LBA4404 for plant transformation (Thomas et al., 1989). Agrobacteriuminfected calli were selected on MSI medium supplemented with 300 g/mL kanamycin + 500 g/mL cefotaxime in dark (Yau et al., 2008). Putatively transformed callus clumps (Fig. 2B) were used for evaluating regeneration on the three regeneration media described below. Regeneration medium: MS-based regeneration medium, MSIII, was the same as that for seed germination and was prepared as described above. B5-based regeneration medium was prepared according to the protocol reported by Gamborg et al. (1976). To obtain MSIII+B5 regeneration medium, equal volumes of MSIII and B5 liquid media were mixed. 1% (w/v) agar was added to the liquid medium to solidify the medium. Regeneration of putatively genetic-transformed callus: Four putatively transformed calli, 3 mm in diameter and derived from a single callus, were evenly distributed on each plate. Twenty plates for each medium were labeled and randomly placed on iron shelves (60 120 cm) with constant uorescent lighting; subculturing to the same medium was performed every 3 weeks. To provide space for continued plantlet growth, regenerated plantlets reaching 5 mm in length were removed from the callus to freshly prepared plates for continued growth during subculturing. The numbers and lengths of plantlets were determined after 6 subcultures, when few new regenerants were observed from the calli. The length from the stem above the tap root to the tip of the leaves of each healthy plant was measured as an index of regeneration ability after 4.5 months of growth. Healthy plants are defined as regenerated plants with green leaves, normal stems and normal roots. Plants were grouped into ve length categories: 0.5-1, 1-2, 3-4, 5-6 and 7-9 cm. Besides, the numbers of regenerants after 2-cm dramatically dropped from MSIII plates or MSIII+B5 plates-some with only 1 or 0 plants. For each medium, the number of plantlets in each length category was recorded. GUS histochemical assay: Histochemical staining of carrot leaf tissues was performed according to Jefferson et al. (1987). Leaf tissues were harvested and vacuum-ltered for 10 min in 5-bromo-4-chloro-3-indoxyl--D-glucuronide (X-gluc) (Gold BioTechnology, Inc., St. Louis, MO, USA) staining solution and then stained overnight at 37C. To check GUS staining, leaves were rst de-chlorophylled by repeated washing in 70% ethanol until all the tissue was bleached.

callus induction (Simon et al. 1990). The number and size of the regenerated plantlets on these three different regeneration media were compared. The presence of the transgene in the regenerated plants from transformed calli was also characterized by GUS staining assay and Southern blotting.

Materials and methods

Experimental procedures to obtain transgenic callus: To investigate the recgeneration ability, putative transgenic B493 callus was used (not the non-transgenic material) because we thought that this would accurately simulate the conditions for producing transgenic carrotincluding the selection of Agrobacterium-infected callus on a medium with a selective agent for several months. Callus induction from explants: Seeds of carrot inbred line B493 were wrapped in two layers of cheese cloth, and the seed surface was sterilized by treatment with 70% (v/v) ethanol for 2 minutes at room temperature, then with 5% (w/v) sodium hypochlorite (NaOCl) containing 0.02% (w/v) Triton X-100 for 15 minutes. After sterilization, seeds were rinsed several times with sterile water from a Milli TM-Q UF Plus Water System (Millipore Corporation, Bedford, MA, USA) and then placed on solid MS (Murashige and Skoog salt formulation) medium supplemented with 3% (w/v) sucrose, 1 g/mL thiamin (B1), 0.1% (w/v) myoinositol (Sigma, St. Louis, MO, USA) and 1% (w/v) agar (Bactoagar, Detroit, MI, USA) and adjusted to pH 5.8 with 0.5N KOH, termed as MSIII. The medium was autoclaved at 121oC and 1.2 kgs/cm2 for 15 minutes and then cooled for plating into 100 15 mm sterile polystyrene petri-dishes (Fisher Scientic, Pittsburgh, PA, USA). Each plate contained 6 seeds. Seeds were germinated on plates and grown under uorescent light. Callus induction medium, termed MSI [MSIII medium supplemented with 1 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) (Sigma, St. Louis, MO, USA) and 21.5 g/L kinetin (Aldrich, Milwaukee, WI, USA)], was used for callus induction. Under sterile conditions, roots of the plantlets were cut into 5 mm lengths and placed on the surface of the MSI medium. Plates were incubated in the dark at room temperature for callus induction (Fig. 2A). Agrobacterium-mediated genetic transformation: Induced calli were then used for genetic transformation. Carrot callus transformation method described by Wurtele and Bulka (1989) was followed. Transformation vector pBI121 (Fig. 1) which comprises T-DNA right border-Nos promoter-nptII gene-Nos

Fig. 1. T-DNA construct of binary vector pBI121 used for Agrobacterium-mediated genetic transformation of carrot callus. Probe used for Southern hybridization was indicated with a bar (diagram is not drawn to scale).

Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium Southern hybridization: Southern hybridization was carried out according to the standard protocol described by Sambrook et al. (1989). Mature carrot plant leaves were collected, lyophilized for two days and used for total genomic DNA isolation with 2x CTAB extraction solution (Murray and Thompson, 1980). Genomic DNA of 5 g was digested with BbuI (isoschizomer of SphI) restriction enzyme overnight at 37C. Digested DNA was run on a 0.8% TAE gel and transferred to a membrane for Southern hybridization according to Yau et. al. (2008). Hybridization was performed with a nptII gene-derived P32-labelled probe which was produced according to the protocol described by Yau et al. (2008). Statistical analysis: To determine whether the regeneration of the plantlets was inuenced by the medium, statistical tests were performed to compare the number of plantlets on one medium to the number of plantlets on another. Since the data consisted of discrete counts, a Poisson distribution (Mendes et al., 1999) was used to model the number of plantlets produced on a given medium in a given length category. The following null hypotheses were tested: Ho: B5 = MSIII; Ho: B5 = MSIII+B5; Ho: MSIII = MSIII+B5 where, B5, MSIII, and MSIII+B5 denote the population mean of the Poisson distribution for each of the three mediums. Each null hypothesis was tested against the two-sided alternative hypothesis that the two means were not equal. The three tests mentioned above were performed for all plantlets (irrespective of length category). Likewise, the three tests were performed separately to compare the media for each length category. It bears mentioning that this test is exact, in the sense that it does not depend upon large sample size asymptotics to provide accurate P-values. The probabilities for the P-values were calculated using S-PLUS (Venables and Ripley, 1997).

better medium than MS-based medium, MSIII, or MSIII+B5 for carrot callus regeneration. B5-based medium was also reported to be a better callus-inducing medium earlier (Hardegger and Sturm, 1998). Taken together, B5-based medium should be used routinely in carrot genetic transformation to improve its efciency. Further analysis may be required if other genotypes of carrot are utilized for genetic transformation. To check the transgenic nature of the regenerated plants, two plants were randomly chosen for a GUS histochemical assay and Southern hybridization analysis. For histochmical assay for GUS activity, the transgenic leaf tissue showed GUS staining with blue color, while the non-transgenic leaf tissue showed no GUS staining (Fig. 2D and 2E). For Southern analysis, genomic DNA of transgenic plants was digested with BbuI and hybridized with a nptII gene-derived P32-labelled probe. Identical hybridization banding pattern was observed for the two transgenic plants, and conrmed that these two lines are siblings which derived from a single callus (Fig. 2F lanes 1 and 2). The wild type (nontransformed) plant exhibited no hybridization signal (Fig. 2F lane 3). Both results from GUS assay and Southern analysis suggest that the T-DNA is present in the transgenic lines. Since the plant lines were derived from the same callus, the signal patterns from Southern analysis showed the same.

Results and discussion

More regenerated plants (432) were obtained from the calli regenerating on B5 regeneration medium (Fig. 2C) as compared to MSIII (24) and MSIII+B5 (28) (Table 1). Each length category for B5 regeneration medium had 41 or more healthy, regenerated plantlets. Regenerated plantlets 5.0-6.0 cm long were the most numerous (175), followed by plantlets with lengths between 3.0-4.0 cm (94 plantlets) (Table 1). In contrast, few carrot plantlets were regenerated on MSIII or MSIII+B5 regeneration medium and no plantlets over 7.0 cm were observed. The overall tests which compared media irrespective of plantlet length demonstrated that B5 produced signicantly more plantlets than MSIII (P < 0.000001). Likewise, B5 produced signicantly more plantlets than MSIII+B5 (P < 0.000001). There was no signicant difference between MSIII and MSIII+B5 (P-value: 0.678). These results demonstrated that B5-based regeneration medium is a
Table 1. Total number and length of regenerated plants from B493 calli on B5, MSIII and MSIII+B5 media Medium Length (cm) Total 0.5-1.0 1.0-2.0 3.0-4.0 5.0-6.0 7.0-9.0 number B5 41a 70a 94a 175a 52a 432 b b b b MSIII 16 6 1 1 0b 24 MSIII + B5 12b 8b 8b 0b 0b 28 Within same column, numbers with the same superscript are not signicantly different at P=0.05.

Fig. 2. Regeneration and characherization of carrot plantlets regenerated from putative Agrobacterium-transformed callus. (A) Soft and yellowish callus induced from carrot seedling hypocotyls (see the representative one by an arrow), (B) Putative transformed callus clumps (light color) emerge from old callus (dark color) under antibiotic kanamycin selection (see the representative one by an arrow), (C) A regenerated plantlet on B5 medium, (D-E) GUS staining of non-transgenic control (D) and transgenic (E) plants, (F) Southern analysis of two transgenic plants (lanes 1 and 2) and a non-transgenic plant (lane 3).

Acknowledgements
We thank Dr. Phil Simon for providing carrot materials for this experiment, and Dr. Roger Thilmony and Dr. Ludmila Tyler for carefully reading the manuscript and giving suggestions. Authors also wish to thank Landon Sego for statistical analysis. Research was funded by the Fresh Carrot Board (N328).

Increased regeneration ability of transgenic carrot callus on B5-based regeneration medium

Shao, C.Y., E. Russinova, A. Iantcheva, A. Atanassov, A. McCormac, D.F. Chen, M.C. Elliott and A. Slater, 2000. Rapid transformation and regeneration of alfalfa (Medicago falcata L.) via direct somatic embryogenesis. Plant Growth Regul., 31: 155-166. Shin, D.H., J.S. Kim, I.J. Kim, J. Yang, S.K. Oh, G.C. Chung and K.H. Han, 2000. A shoot regeneration protocol effective on diverse genotpypes of sunower (Helianthus annuus L.). In Vitro Cell. Dev. Biol.-Plant, 36: 273-278. Simon, P.W. 1997. Plant pigments for color and nutrition. HortScience, 32: 12-13. Simon, P.W., C.E. Peterson and W.H. Gabelman, 1990. B493 and B9304, carrot inbreds for use in breeding, genetics, and tissue culture. HortScience, 25: 815. Simon, P.W., X.Y. Wolff, C.E. Peterson, D.S. Kammerlohr, V.E. Rubatzky, J.O. Strandberg, M.J. Bassett and J.M. White, 1989. High carotene mass carrot population. HortScience, 24: 174-175. utar-Vozli , J., B. Javornik and B. Bohanec, 1999. Studies of somaclonal variation in hop (Humulus lupulus L.). Phyton (Austria), 39: 283-287. Thomas, J.C., M.J. Guiltinan, S. Bustos, T. Thomas and C. Nessler, 1989. Carrot (Daucus carota L.) hypocotyl transformation using Agrobacterium tumefaciens. Plant Cell Rep., 8: 354-357. Venables, W.N. and B.D. Ripley, 1997. Modern Applied Statistics with S-PLUS. Second Edition, Springer. Winicov, I. 1996. Characterization of rice (Oryza sativa L.) plants regenerated from salt-tolerant cell lines. Plant Sci., 113: 105-111. Wurtele, E. and K. Bulka, 1989. A simple, efcient method for the Agrobacterium-mediated transformation of carrot callus cells. Plant Sci., 61: 253-262. Yang, Y.S., Y.D. Zheng, Y.L. Chen and Y.Y. Jian, 1999. Improvement of plant regeneration from long-term cultured calluses of Taipei 309, a model rice variety in in vitro studies. Plant Cell Tiss. Org. Cult., 57: 199-206. Yau, Y.Y., S.J. Davis, A. Ipek and P.W. Simon, 2008. Early identication of stable transformation events by combined use of antibiotic selection and vital detection of green uorescent protein (GFP) in carrot (Daucus carota L.) callus. Agricultural Sciences in China, 7: 664-671. Yau, Y.Y. and P.W. Simon, 2005. Molecular tagging and selection for sugar type in carrot roots using co-dominant, PCR-based markers. Mol Breed., 16: 1-10. Yin, Y., S. Li, Y. Chen, H. Guo, W. Tian, Y. Chen and L. Li, 1993. Fertile plants regenerated from suspension culture-derived protoplasts of an indica type rice (Oryza sativa L.). Plant Cell Tiss. Org. Cult., 32: 61-68.
Received: November, 2011; Revised: December, 2011; Accepted: January, 2012

References
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Journal

Journal of Applied Horticulture, 14(1): 7-12, 2012

Appl

Avoiding the use of plant growth regulator in geranium production by application of a cyclic decit irrigation strategy
Patrick Riga Department of Plant Production and Protection, Basque Institute of Agricultural Research and Development (NEIKERTecnalia), Parque Tecnolgico de Bizkaia, P. 812, E-48160 Derio, Spain. E-mail: priga@neiker.net

Abstract
Plant growth regulators (PGRs) are commonly used in ornamental plant production to improve the decorative value of the plants and to meet marketable targets. The PGRs mostly used in ornamental plant culture are chemical growth retardants that control the size of plants, improve compactness and enhance owering. However, the use of PGRs has been restricted under current legislation, and modied culture practices should be implemented to produce the desired quality of plants. Ornamental plant quality traits are determined by the genetic background of the plant and environmental conditions such as water availability. In the present study, the responses of growth and ower production in geranium (Pelargonium peltatum L.) subjected to cyclic decit irrigation (CDI) were characterized to evaluate the technique as an alternative to the application of a plant growth regulator (daminozide). The leaf water potential of plants under CDI was lower than in control and PGR-treated plants. Moreover, the aerial dry mass, stem dry mass, leaf number, leaf blade area, specic leaf area and stem number of plants under CDI and PGR-treated plants were similar. However, the percentage of plants with at least one opened ower and the number of inorescences per plant were increased by CDI. The marketable quality of the plants subjected to CDI was higher than that of the PGR-treated plants. Moreover, the water use efciency of plants under CDI was 21% higher than that of PGR-treated plants, leading to a 10% reduction in the total water consumption during production. Key words: Daminozide, Pelargonium peltatum, water stress.

Introduction
Chemical growth regulators are commonly used in potted plant production to control shoot elongation and to meet the quality criteria of the markets (Lodeta et al., 2010; Baon et al., 2009; Krause et al., 2003). Daminozide (N-dimethylamino succinamic acid) is used on a variety of ornamentals to produce more compact plants. The compound is absorbed by the leaves and translocated throughout the plant (Moore, 1968). It acts by inhibiting synthesis of gibberellic acid (mainly GA1 and GA8) in the plant (Brown et al., 1997), thereby controlling excessive growth. The inhibitory effect of daminozide is greatest immediately upon application, and the effect becomes less pronounced thereafter, therefore continued growth regulation is accomplished by reapplication of the compound every 10 to 14 days (Rothenberger, 1964). Daminozide must therefore be applied more than once in order to produce a good level of growth retardation in most pot plants. Although daminozide is relatively non-toxic to mammals (the oral LD50 in rats is 8400 mg kg-1, the dermal LD50 in rabbits is > 1600 mg kg-1 and the inhalation LC50 in rabbits is > 147 mg L-1; Meister, 1992), the principal health concern related to use of daminozide is the carcinogenic potential of unsymmetrical dimethyl hydrazine (UDMH), a contaminant and a metabolite of daminozide. UDMH is formed in the body during food processing, or when spray mixes containing daminozide are left standing in the mixing tank. Commercial daminozide contains 0.005% UDMH (US EPA, 1992). A metabolic study in swine showed that 1% of ingested daminozide is converted to UDMH. The US Environmental Protection Agency (1992) estimates that 0.012% of a daminozide solution converts to UDMH when allowed to stand in a tank for 24 hours. In female rats supplied

with UDMH in their drinking water at concentrations of 0, 1, 50 or 100 ppm for 2 years, there was a signicant dose-related increase in liver tumors (US EPA, 1992). All use of daminozide on food crops was voluntarily cancelled by the manufacturer in 1989 and the product is currently registered only for use on ornamental and bedding plants. However, given the potential toxicity of the metabolite, alternative techniques for controlling plant growth are required. As plants grow, most of the increased size and weight is due to increased water content (Kramer and Boyer, 1995), and it is well known that water stress inuences plant growth at various levels, ranging from cell to plant parts. When water is limiting, a number of plant functions are inhibited. Growth is one of the most water-sensitive physiological processes and leaf growth is one of the rst types of growth to decrease, before photosynthesis is affected (Boyer, 1970). The above-mentioned concept has been applied in agriculture for about twenty years, in the form of novel methods of irrigation scheduling such as keeping plants under a slight water decit, the so-called regulated decit irrigation technique (Chalmers et al., 1986) or supplying irrigation alternately to different parts of the root system, leading to the partial root-zone drying technique (Dry and Loveys, 1998). Two main techniques have been used to control plant growth in the production of greenhouse ornamentals: regulated decit irrigation (Van Iersel et al., 2010; lvarez et al., 2009; Blanusa et al., 2009) and cyclic decit irrigation (Niu et al., 2007; Hansen and Petersen, 2004; Petersen and Hansen, 2003), in which, the water content of the growing medium alternates between container capacity and a very low soil water potential, sometimes leading to visible signs of wilting.

Geranium production by application of cyclic decit irrigation the substrate water potential at which fertigation started. A typical time course of substrate water potential is reported elsewhere (Riga et al., 2003). Plants subjected to cyclic decit irrigation did not show any visible signs of wilting. Each treatment was applied to 56 plants. Water retention properties of the substrate: The moisture characteristics of the substrate were determined using a tension table apparatus (De Boodt and Verdonck, 1972; Jamison, 1958) for water potentials between 0 and -10 kPa. The substrate water content at -15 kPa was estimated using van Genuchtens model (1980), as follows: where, s is the substrate water potential, is the water content, s and r are the saturated and the residual water content respectively, , n, and m are dimensionless empirical hydraulic parameters. r was measured after drying the substrate at room temperature for a week (r = 4%). The van Genuchtens model was tted to observed data by minimizing the sum of quadratic differences between observed and calculated data by iterative Newton-Raphson optimization. The substrate water retention data and tted curve are shown in Fig. 2. The estimated value of the substrate water content corresponding to a water potential of -15 kPa was 43.62%. Measured crop parameters: In order to determine biomass production, the plants (from n = 6 to n = 18) were harvested randomly at 30, 50 and 75 days after treatment. Dry weights were measured after drying fresh biomass at 70 C to constant weight (48 h). Leaves were counted and leaf area was measured as follows: harvested leaves were immediately photocopied (Xerox, model XD332), and images were scanned to count the number of total black pixels with an image analyser. The relationship between the total number of black pixels and the corresponding area was calculated from a pre-established calibration curve. The specic leaf area (SLA) was calculated by dividing total leaf area (cm2) by total leaf dry weight (g). Total water consumption was measured weekly by weighing the tanks containing the nutrient solution. Water use efciency of the culture was measured as dry weight produced per unit mass of water loss by evapotranspiration. Marketable quality was evaluated by the number of open owers s=

The objective of the present study was to characterize growth and ower production in geranium plants under cyclic decit irrigation, which is a possible alternative to the application of plant growth regulators, and to evaluate any environmental advantages of the technique.

Materials and methods

Crop conditions: Individual rooted cuttings of Pelargonium peltatum L. were transplanted to 1 L (diameter, 13.0 cm; height, 11.4 cm) containers filled with a commercial peat substrate (Floragard, KTS2, long fibres) and grown in an automated polycarbonate-covered greenhouse located at The Basque Institute of Agricultural Research and Development (NEIKER, A.B., Biscay, Spain, latitude 43 17 N, longitude 2 52 W, altitude 77 m). The pots were placed on benches at a density of 15 plants m-2. Plants were fertigated by sub-irrigation with a nutrient solution containing a commercial fertilizer (3N-2P2O5-3K2O-0.4MgO, 6SO3, 0.002B, 0.004Cu, 0.01Fe, 0.01Mn, 0.0002Mo, 0.002Zn). The electrical conductivity (EC) of the nutrient solution was 0.5 mS cm-1, and the pH was adjusted to 5.0 with phosphoric acid. The duration of the fertigation treatments and their frequency were controlled by tensiometers (Lapton Control de Riego, Bizkaia, Spain) inserted into the middle of one container per treatment and connected to an automatic pump control system. Each replicate had its own independent tank (50 L) containing nutrient solution and a pump controlled by the tensiometers. The air-heating was set to 10 and 12 C night/day, and vent opening temperatures were 12 and 15 C night/day, respectively. The climate data measured inside the greenhouse are shown in Fig. 1. Treatments: Ten days after planting, the plants were subjected to one of three treatments: i) fertigation was started when the tensiometer reading reached a substrate water potential equal to -5 kPa (control); ii) plants were fertigated when the water potential of the substrate reached -5 kPa and were sprayed (1000 L ha-1) three times (every 15 days) with a 0.5% solution of daminozide (N-dimethylamino succinamic acid) (PGR-treatment), or iii), plants were fertigated when the water potential of the substrate reached -15 kPa (cyclic decit irrigation treatment). Daminozide (B-nine) was purchased in powdered form (85.0% ww-1 active ingredient) from Uniroyal Chemical Co., Middlebury, CT, USA. In all treatments, fertigation ceased when the substrate water potential reached saturation. The water content of the growing medium alternated between container capacity and the value of

[((s- r)/ (- r))1/m -1]1/n

Fig. 1. Mean daily temperature (open circles) and relative humidity (closed squares) recorded inside the greenhouse.

Fig. 2. Substrate water retention data obtained by the tension table technique, and curve tted with Van Genuchtens model. n, m and alpha are empirical parameters. Means SD, (n = 3). When no bar is shown, it is included in the width of the symbol.

Geranium production by application of cyclic decit irrigation and by visual estimation of the global plant quality on the basis of local market requirements. Leaf water status: Mid-day leaf water potential was measured between 1300 and 1500 (local time) using a pressure chamber (PMS Instrument Company, Oregon, USA) on the uppermost, fully expanded leaves of 5 plants per treatment (one leaf per plant). Cut leaves were immediately enclosed in plastic bags to prevent additional evaporation during handling of samples. Statistical analysis: The data were analysed by the GLM and REG procedures in SAS (version 8.0. Differences between means were analysed by the Duncans multiple range test.

Results
Leaf water potential: The mid-day leaf water potential was lower in plants under cyclic decit irrigation (-15 kPa) than in control plants (-5 kPa) and PGR-treated plants (-5 kPa + PGR) (Fig. 3). Compared to control plants, the PGR treatment had no effect on L.
Fig. 4. Changes in total aerial dry weight over 75 days after the beginning of the treatments. Plants were irrigated when the tensiometer reading reached -5kPa (control) or -15kPa (water decit). In addition, some plants irrigated at 5kPa were sprayed every 15 days with a plant growth regulator (-5kPa + PGR). Means SD. Means followed by the same letters are not signicantly different at P < 0.05. ns = not signicant.

to PGR-treated plants (Table 1). For the same leaf area per plant, if the number of leaves increases depending on the treatment, then the leaf area per leaf decreases (Table 1). In addition, the SLA was lowest in plants under cyclic water decit. There was a signicant difference in SLA between the water decit treatment and the control treatment. Stem dry weights were only affected by treatments after 75 days (Fig. 6). The dry weight of control plants was signicantly higher than that of PGR-treated plants. Stem dry weights represented about 10% of the value of total aerial dry mass over the period of the experiment (Fig. 4 and 6) and were not affected by treatments. However, 75 days after the start of treatment, the number of stems per plant was signicantly higher in both cyclic water decit and PGR-treated plants, than in control plants (Table 2). Nevertheless, there were no signicant differences in the number of stems between the -15 kPa and PGR treatments. Moreover, the internode lengths were signicantly lower in plants under cyclic

Fig. 3. Effect of treatments on midday leaf water potential measured between 1300 and 1500 (local time) on the uppermost, fully expanded leaves of 5 plants per treatment (one leaf per plant). Means SD. Means followed by the same letters are not signicantly different at P < 0.05.

Plant growth parameters: Total aerial dry weights (leaves + stems) were affected by treatments after 75 days only (Fig. 4). The dry weight of control plants was signicantly higher than that of the PGR-treated plants, conrming the positive effect of the daminozide treatment in reducing plant growth. The treatments did not affect the leaf area per plant over the period of the experiment (Fig. 5). Nevertheless, 75 days after the start of treatments the plants under cyclic water decit had more leaves than the control plants, but with no difference compared
Table 1. Effect of treatments on leaf number and leaf blade area at the end of the experiment (75 days) SLA Treatment Leaves Leaf blade area (cm2 g-1) number (cm2) -5 kPa 30.00 (5.46) a 27.37 (2.10) a 3.77 (0.53) a -5 kPa + PGR 31.36 (3.75) ab 23.35 (1.70) ab 3.15 (0.56) ab -15 kPa 34.50 (4.62) b 21.96 (1.99) b 2.73 (0.60) b SLA = specific leaf area. Values are means for 6 plants. Standard deviations are shown in bracket. Means followed by the same letters are not signicantly different at P < 0.05.

Fig. 5. Change in leaf area over 75 days after the beginning of the treatments. Plants were irrigated when the tensiometer reading reached 5kPa (control) or -15kPa (water decit). In addition, some plants irrigated at 5kPa were sprayed every 15 days with a plant growth regulator (5kPa + PGR). Means SD. Means followed by the same letters are not signicantly different at P < 0.05. ns = not signicant.

10

Geranium production by application of cyclic decit irrigation

Table 2. Effect of treatments on stem number and on internode length at the end of the experiment (75 days) Treatment Stem number Internodal length (cm) -5 kPa 2.76 (0.63) a 3.76 (0.48) a -5 kPa + PGR 3.21 (0.65) b 2.85 (0.46) b -15 kPa 3.46 (0.68) b 3.22 (0.34) c Values are means for 13 plants. Standard deviations are shown in brackets. Means followed by the same letters are not significantly different at P < 0.05. Table 3. Effect of treatments on percentage of plants with at least one opened ower (OF) and on number of inorescences per plant (IP) at the end of the experiment (75 days) Treatment OF (%) Number of IP -5 kPa 45.1 (5.2) a 2.12 (0.04) a -5 kPa + PGR 44.9 (4.5) a 2.36 (0.17) ab -15 kPa 62.9 (6.2) b 2.50 (0.09) b Values are means for 13 plants. Standard deviations are shown in brackets. Means followed by the same letters are not significantly different at P < 0.05.

Fig. 6. Changes in stem dry weight per plant over 75 days after the beginning of the treatments. Plants were irrigated when the tensiometer reading reached -5kPa (control) or -15kPa (water decit). In addition, some plants irrigated at 5kPa were sprayed every 15 days with a plant growth regulator (-5kPa + PGR). Means SD. Means followed by the same letters are not signicantly different at P < 0.05. ns = not signicant.

there was no difference between water stressed and PGR-treated plants. Total evaporation in the plants subjected to cyclic water decit or PGR treatment was respectively 32 and 23% lower than in control plants. Plant under water decit showed signicant higher WUE values (expressed as the ratio of production of aerial biomass against total evapotranspiration (Jones, 2004) than control and PGRtreated plants (Fig. 8b). The WUE values were 21 and 27% higher

Fig. 7. Changes in daily evapotranspiration per plant over 75 days after the beginning of the treatments. Plants were irrigated when the tensiometer reading reached -5kPa (control) or -15kPa (water decit). In addition, some plants irrigated at 5kPa were sprayed every 15 days with a plant growth regulator (-5kPa + PGR). Means SD. Means followed by the same letters are not signicantly different at P < 0.05. ns = not signicant.

water decit and treated with PGR than in control plants (Table 2). The internodal length was longest in control plants and shortest in PGR-treated plants. Water consumption: Daily evapotranspiration of potted plants was affected early on by treatments (Fig. 7). At 30 days after the start of treatment, plants under cyclic water decit showed the lowest evapotranspiration values, about two times lower than in control and PGR-treated plants. At this time, PGR treatment did not yet have any effect on evapotranspiration, in comparison with control plants. From day 50, evapotranspiration was lower in PGR-treated plants than in control plants and higher than in water stressed plants. After 75 days, signicant differences were found only between control plants and the other two treatments. Thus, the highest value of total evapotranspiration over the whole period of the experiment was observed in control plants (Fig. 8a), whereas,

Fig. 8. Effects of treatments on total evapotranspiration and water use efciency (WUE) after 75 days. Means SD. Means followed by the same letters are not signicantly different at P < 0.05. ns = not signicant.

Geranium production by application of cyclic decit irrigation in water stressed plants than in PGR-treated and control plants, respectively. Nevertheless, PGR treatment did not affect the WUE value compared to control plants. Marketable quality: Under cyclic water decit, the number of plants with at least one opened ower at 75 days after the start of treatment was higher than in control and PGR-treated plants (Table 3), and about 20% more of the water stressed plants had one opened ower. In addition, plants under the cyclic water decit treatment had about 34% more inorescences per plant than control plants, and the same number as PGR-treated plants. Treatment with PGR did not affect either the number of plants with at least one opened ower or the number of inorescence per plant. At 75 days after the start of treatment, visual evaluation of the plant quality was carried out on the basis of the local market requirements. Compact plants with at least one opened ower, with four or more ramied branches, and leaves of normal size and colour, were considered as marketable. A large number of inorescences per plant also increased the marketable quality. Plants grown under cyclic decit irrigation and PGR-treated plants displayed higher overall quality than control plants, with no differences between the treatments (data not shown). The control plants were not of marketable quality because of their lack of compactness and large leaves.

11

measured. It is well known that gibberellins regulate growth (by increasing the rate of stem elongation), and as daminozide inhibits synthesis of these hormones (Brown et al., 1997), the main effect of daminozide is to reduce the internode length, resulting in more compact plants. Effects of treatments on water consumption: The daily and total evapotranspiration values were lowest, and the WUE values were highest in the P. peltatum plants under water decit. The main effect of water decit is to reduce the vegetative growth, leaf stomata conductance and transpiration, leading to an increase in the WUE as long as the water decit is not too severe. When water is limited, the productivity of a plant that uses a nite water supply most efciently would be positively affected (Loveys et al., 2004). Plants produced with sufcient water supply had larger leaf areas and consequently these plants displayed the highest water consumptions and the lowest WUE. Interestingly, from 50 days after the start of the daminozide treatment, daily evapotranspiration in the treated plants was lower than in control plants, and after 75 days, the values decreased to the same level as in the water stressed plants, although the total leaf area was the same in the daminozide-treated plants and in the other plants. In addition, total evapotranspiration was lower in daminozidetreated plants than in control plants, but with no difference in the WUE values. Reports on the effect of daminozide on the water plant status are rather limited. However, in chrysanthenum, daminozide treatment was found to affect the stomata index, and the length and width of the stomata, resulting in leaves with xeromorphic properties (Kilic et al., 2009). Effects of treatments on marketable quality: The results of the present study showed that decit irrigation produced the highest percentage of marketable plants and the highest percentage of inorescences in P. peltatum, as previously reported (Riga et al., 2003). In the case of P. hortorum a moderate water decit (midday leaf water potential L -0.7 MPa) did not affect the number of inorescence or the number of open owers per plant relative to control plants, while a severe water decit (L -0.8 MPa) decreased the values of these two parameters (Sanchz-Blanco et al., 2009). In the present study, the water stress conditions resulted in L values between -0.8 and -1.1 MPa, suggesting that P. peltatum is more tolerant to water stress than P. hortorum. Many plant species can be induced to ower by the application of stress, e.g. water stress (Wada and Takeno, 2010). Water stress inuences the number of ower buds and their opening depending on the plant species and the intensity of the stress. In the present study, daminozide treatment did not affect the percentage of plant with at least one opened ower or the number of inorescence per plant in comparison with control plants, as also found in a study on Tagetes patula (Krause et al., 2003). In contrast, daminozide stimulated owering in Petunia hybrida, Impatiens walleriana (Krause et al., 2003) and in Rhododendron sp. (Meijn et al., 2009). The mode of action of daminozide on owering has not yet been elucidated. Use of a cyclic decit irrigation strategy proved to be a good alternative over the use of plant growth regulator for geranium (P. peltatum) production, as it maintained or even improved the marketable value of the plants. In comparison with the application of PGR, this technique reduced water consumption by 10%, and

Discussion
Effects of treatments on leaf water status and plant growth parameters: The midday leaf water potential (L) ranged between -0.8 and -1.1 MPa in plants under cyclic decit irrigation, which is consistent with a previous report of L values between -0.7 and -0.8 MPa for Pelargonium hortorum plants under moderate or severe water stress, respectively (Sanchz-Blanco et al., 2009). Reducing the water supply to plants affects leaf water status, and leaf growth is one of the rst parameters that is limited, before photosynthesis is affected (Boyer, 1970). Under the experimental conditions in the present study, the leaf blade area and SLA values were lowest, and the number of leaves were highest in the water stressed plants. The negative effect of water stress on leaf blade area has also been reported for Hibiscus rosa-sinensis (Hansen and Petersen, 2004), miniature roses (Williams et al., 1999) and P. hortorum (Sanchz-Blanco et al., 2009). However, although the number of leaves was lower in water stressed P. hortorum than in control plants, the water stressed P. peltatum in the present study had more leaves because of a higher value of stem number than well watered plants. In addition, the SLA of water stressed leaves was lower because of a signicant decrease in leaf blade area. Similar SLA response to water stress had been reported for legume species (Villagra and Cavagnaro, 2006). The SLA reects aspects of leaf morphology, such as leaf density and thickness. Leaves tend to be more dense under water decit than under well watered conditions, which leads to a decrease in SLA (Navas and Garnier, 2002; CastroDez et al., 2000). In plants treated with the plant growth regulator daminozide the leaf water potential was similar to that in control plants, the internode length was shorter and stem number was higher; treatment with daminozide did not affect the other leaf parameters

12

Geranium production by application of cyclic decit irrigation

Kilic, S., S. Kazaz and K. Cavusoglu, 2009. Effects of daminoside (Alar 85) treatment on the stomata movements of chrysanthemum (Dendranthema grandiorum, (Ramat.) Kitamura) seedling growth in different day length condition. J. Anim. Vet. Adv., 8: 2013-2017. Kramer, J.K. and J.S. Boyer, 1995. Water Relations of Plants and Soils. Academic Press, INC. California, USA Krause, J., E. Krystiniak and A. Schroeter, 2003. Effect of daminozide on growth and owering of bedding plants. J. Fruit Orn. Plant Res., 11: 107-112. Lodeta, K.B., S.G. Ban, S. Perica, G. Dumicic and L. Bucn, 2010. Response of poinsettia to drench application of growth regulators. J. Food Agr. Env., 8: 297-301. Loveys, B.R., M. Stoll and W.J. Davies, 2004. Physiological approaches to enhance water use efciency in agriculture: exploiting plant signalling in novel irrigation practice. In: Water Use efciency in Plant Biology. M.A. Bacon (Ed.), Blackwell Publishing, Oxford, UK, pp. 114-141. Meijn, M., R. Rodrguez, M.J. Caal and I. Feito, 2009. Improvement of compactness and oral quality in azalea by means of application of plant growth regulators. Sci. Hort., 119: 169-176. Meister, R.T. 1992. Farm Chemicals Handbook 92. Meister Publishing Company, Willoughby, OH. Moore, T.C. 1968. Translocation of the growth retardant N-N dimethylaminosuccinamic acid 14C. Bot. Gaz., 129: 280-285. Navas, M.L. and E. Garnier, 2002. Plasticity of whole plant and leaf traits in Rubia peregrina in response to light, nutrient and water availability. Acta Oecol., 23: 375-383. Niu, G., D.S. Rodrguez, L. Rodrguez and W. Mackay, 2007. Effect of water stress on growth and ower yield of Big Bend bluebonnet. HortTech., 17: 557-560. Petersen, K.K. and C.W. Hansen, 2003. Compact campanula carpatica Uniform without chemical growth. Europ. J. Hort. Sci., 68: 266271. Riga, P., S. lava, A. Usn, F. Blanco, C. Garbisu, A. Aizpurua, T. Tejero and A. Larrea, 2003. Evaluation of recycled rockwool as a component of peat-based mixtures for geranium (Pelargonium peltatum L.) production. J. Hort. Sci. Biotech., 78: 213-218. Rothenberger, R.R. 1964. Studies of the translocation, metabolism and environmental responses of ornamental plants to chemical growth retardants. Ph.D Dissertation, Miss. State Univ., Missouri. Sanchz-Blanco, M.J., S. lvarez, A. Navarro and S. Baon, 2009. Changes in leaf water relations, gas exchange, growth and owering quality in potted geranium plants irrigated with different water regimes. J. Plant Physiol., 166: 467-476. US Environmental Protection Agency. Oct. 8, 1992. Daminozide; notice of nal determination for non-food uses and termination of the daminozide Special Review. Federal Register, 57(196): 46436-44. Van Genuchten, R. 1980. Predicting the hydraulic conductivity of unsaturated soils. Soil Sci. Soc. Am. J., 44: 892-898. Van Iersel, M.W., S. Dove, J.G. Kang and S.E. Burnett, 2010. Growth and water use of petunia as affected by substrate water content and daily light integral. Hortscience, 45: 277-282. Villagra, P.E. and J.B. Cavagnaro, 2006. Water stress effects on the seedling growth of Prosopis argentina and Prosopis alpataco. J. Arid Environ., 64: 390-400. Wada, K.C. and K. Takeno, 2010. Stress-induced owering. Plant Signal. Behav., 8: 944-947. Williams, M.H., E. Rosenqvist and M. Buchhave, 1999. Response of potted miniature roses (Rosa x hybrida) to reduced water availability during production. J. Hort. Sci. Biotech., 74: 301-308.
Received: January, 2011; Revised: July, 2011; Accepted: December, 2011

eliminated the cost of the PGR (about 3500 Euros per ha) and its application. Without the need for daminozide and thus the absence of harmful residues, the working conditions of the growers would improve and risk of environmental contamination would decrease. The decit irrigation strategy could therefore lead to an environmentally safer production of ornamental plants and to healthier working conditions.

Acknowledgements
This research was financially supported by the Department of Environment, Agriculture and Fisheries of the Basque Government. The author thanks Sergio Alava for his skilful technical assistance and Dr Christine Francis for correcting the manuscript.

References
lvarez, S., A. Navarro, S. Baon and M.J. Snchez-Blanco, 2009. Regulated decit irrigation in potted Dianthus plants: Effects of severe and moderate water stress on growth and physiological responses. Sci. Hort., 122: 579-585. Baon, S., J. Miralles, A. Navarro and M.J. Snchez-Blanco, 2009. Inuence of paclobutrazol and substrate on daily evapotranspiration of potted geranium. Sci. Hort., 122: 572-578. Blanusa, T., E. Vysini and R.W.F. Cameron, 2009. Growth and owering of petunia and impatiens: effects of competition and reduced water content within a container. Hortscience, 44: 1302-1307. Boyer, J.S. 1970. Leaf enlargement and metabolic rates in corn, soybean, and sunower at various leaf water potentials. Plant Physiol., 46: 233-235. Brown, R.G.S., H. Kawaide, H. Yang, Y.Y. Radmacher and W. Kamiya, 1997. Daminozide and prohexadione have similar modes of action as inhibitors of the late stages of gibberellin metabolism. Physiol. Plant., 101: 309-313. Castro-Dez, P., J.P. Puyravaud and J.H.C. Cornelissen, 2000. Leaf structure and anatomy as related to leaf mass per area variation in seedlings of a wide range of woody plant species and types. Oecologia, 124: 476-486. Chalmers, D.J., G. Burge, P.H. Jerie and P.D. Mitchell, 1986. The mechanism of regulation of Bartlett pear fruit and vegetative growth by irrigation withholding and regulated decit irrigation. J. Am. Soc. Hort. Sci., 111: 904-907. De Boodt, M. and O. Verdonck, 1972. The physical properties of the substrates in horticulture. Acta Hort., 26: 37-44. Dry, P.R. and B.R. Loveys, 1998. Factors inuencing grapevine vigour and the potential for control with partial rootzone drying. Aus. J. Grape Wine Res., 4: 140-148. Hansen, C.W. and K.K. Petersen, 2004. Reduced nutrient and water availability to hibiscus rosa-sinensis Cairo Red as a method to regulate growth and improve post-production. Europ. J. Hort. Sci., 69, S.: 159-166. Jamison, V.C. 1958. Sand-silt suction column for determination of moisture retention. Soil Sci. Soc. Am. Proc., 22: 82-83. Jones, H. 2004. What is water use efciency? In: Water Use Efciency in Plant Biology. Bacon, M.A. (Ed.), Blackwell Publishing, Oxford, UK, pp. 7-41.

Journal

Journal of Applied Horticulture, 14(1): 13-17, 2012

Appl

Effect of light emitting diodes (LEDs) on postharvest needle retention of balsam r (Abies balsamea L.)
R. Scott Veitch, Rajasekaran R. Lada* and Mason T. MacDonald Nova Scotia Agricultural College, Department of Environmental Science, P. O. Box 550, B2N5E3, Bible Hill, Nova Scotia, Canada. *E-mail: rlada@nsac.ca

Abstract
Two experiments were conducted to understand the effect of light emitting diodes on postharvest abscission in balsam r (Abies balsamea L.) branches. In one experiment, branches were pre-exposed to the uorescent light, LEDs, or darkness for 1, 4, 8, 12, 24, or 48 h. In a second experiment, branches were constantly exposed to uorescent lights, LEDs, or darkness. The response variable was needle retention duration (NRD). A 48-hour exposure time to red, white, or blue LEDs signicantly (P < 0.001) increased NRD by approximately 75, 118, or 127%, respectively, compared to a cool white uorescent lighting or darkness. Constant exposure to any LED signicantly (P < 0.001) improved NRD compared to uorescent lights or darkness, though white and red LEDs were most effective. It is speculated that LED-promoted needle retention could possibly be due to changes in carbohydrate synthesis similar to those observed during cold acclimation. Key words: Abscission, Abies balsamea, balsam r, conifer, light emitting diode, needle retention, postharvest, senescence

Introduction
Balsam r is the principal Christmas tree species and specialty horticultural product grown in Nova Scotia. Balsam fir are grown on over 10,000 ha (25,000 ac) and approximately 1.5 to 2.0 million trees are harvested each year. Overall, the industry generates 72 million dollars annually in the Atlantic region and employs several hundred people (CTCNS, 2010). Postharvest needle drop is a signicant challenge faced by the Christmas tree industry of Atlantic Canada. In recent years, the severity of needle loss has escalated to an extent that threatens the survival of Christmas tree and greenery industry, where the number of annual sales is starting to decrease (Chastagner and Benson, 2000). While needle losses occur during harvesting, handling, transportation, and at display stands to a certain extent, extensive needle loss after consumers purchase has become a matter of great concern for the industry (Mitcham-Butler et al., 1988). The industry suffers huge economic losses due to the reduced marketability, as consumers no longer tolerate needle loss and there is an increasing trend towards purchasing articial trees. While the exact reason for needle drop is yet to be determined, it is commonly believed that increased demand from foreign markets requires earlier harvesting. Harvesting balsam r often begins in early October in Nova Scotia, which results in poor needle retention (MacDonald et al., 2010a; MacDonald and Lada, 2008). In addition, warmer fall temperatures in late October have reduced the needle retention capabilities of Christmas trees worldwide (Chastagner and Riley, 2003). The use of LEDs in horticulture was originally presented as a potential technology for space-based plant research chambers or bioregenerative life support systems (Bula et al., 1991; Barta et al., 1992). Certain advantages that LEDs provide, such as small size, durability, long operational life, wavelength specicity, and relatively cool emitting surface, could be useful during

transport and storage of Christmas trees (Li et al., 2010). In addition, LEDs are considered eco-friendly due to their low electricity requirements and long operational lifespan. Light spectra quality, intensity and duration at different wavelengths, particularly those at red or blue, inuence plants by triggering physiological reactions including dormancy, photoperiodism, owering, senescence, and abscission (Li et al., 2010, Okamoto et al., 1996; Tennessen et al., 1993; Yanagi et al., 1996). For example, red light is important in the development of the photosynthetic apparatus and starch accumulation (Saebo et al., 1995) while blue light is important in development of chlorophyll, chloroplast development, and enzyme synthesis (Senger, 1982). Light controls hypocotyls growth and activity of enzymes associated with nitrogen metabolism in Scots pine trees using a combination of phytochrome and blue/ultra-violet light. Other studies done on Scots pine trees showed that far red and red light invoke phytochrome system and induce cold hardening (Beck et al., 2004). Leaf senescence and abscission are affected by light. Leaves kept in the dark senesce faster compared to those exposed to light (van Lieburg et al., 1990). In contrast, certain spectra of light can delay abscission. For example, abscission resistance in mung bean leaves is enhanced with red light (Curtis, 1978) while red LEDs delayed postharvest senescence and abscission in ornamental owers such as hibiscus and lillies (van Lieburg et al., 1990; van Meeteren and van Gelder, 2000). However, there is no information relating the role of LEDs to postharvest abscission in balsam r. The objective of this study was to understand the effect of certain spectrums (blue, red and white) of LEDs on needle abscission in postharvest balsam r.

Materials and methods

Sample collection: Balsam fir branches containing the two most recent years of growth were collected from mature grafted

14

Effect of LEDs on postharvest balsam r needle retention

Table 1. Summary of light sources used in this experiment. Light intensity is reported as the mean from six measurements standard deviation Treatment Fluorescent* Blue Red White** Dark Peak wavelength (nm) 400 - 800 465 650 465, 575, 650 N/A Light intensity (mol s-1 m-2) 253.7 4.5 17.0 0.6 17.0 0.6 20.0 0.7 N/A

trees (approximately 17 years old) at the Tree Breeding Center, Department of Natural Resources, Debert, Nova Scotia, Canada (45 25 N, 63 28 W). A total of 112 trees were randomly selected for experiment 1 on August 11, 2009; another 28 trees were randomly selected for experiment 2 on September 24, 2009. In each experiment, one branch was cut from each tree from the south facing side at between 1.0 to 1.5 m above ground to serve as a sample. Cut branches were placed in distilled water and transported to a growth chamber with a day/night temperature regime of 18/10C and 60% relative humidity. Light was provided using uorescent lights at an intensity of 254 mol m-2 s-1 for 16 hours each day. All branches were given a fresh cut 3 cm from the stem and weighed before they were placed in 250 mL ask of distilled water. Short-term exposure to LEDs: Separate, but similar, experiments were conducted to determine the effect of short-term exposure to blue LEDs, red LEDs, white LEDs (LED wholesalers.com Burlingame, California, USA), uorescent light, or darkness on postharvest needle abscission. Each experiment followed a completely randomized design using exposure duration (0, 1, 4, 8, 12, 24, or 48 hours) as a treatment. In each treatment, a branch was placed in a custom-built chamber and exposed to certain lighting or darkness for a specied length of time and then placed in growth chamber conditions (described above). This procedure was repeated 4 times for each exposure time, each with a separate branch. Fluorescent light and dark treatments each served as control, as they each simulate environments in storage and shipping. LED treatments involved exposure to a 30 x 30 cm LED panel. Light intensity in each chamber was measured using the LI-188B Integrating Quantum Photometer (LI-COR, Lincoln, NE, USA) and reported as the average of six measurements from random locations in each chamber (Table 1). Air ow was supplied to each chamber by forcing air into the bottoms of the chambers at a rate of 3 L min-1 using an Elite 802 air pump (Hagen, Truro, NS, Canada). The internal temperature of all LED chambers was monitored throughout the experiment and was consistent with growth chamber temperatures. The response variable used was needle retention duration (NRD) dened by MacDonald et al. (2010a; 2010b), as the length of time to lose 50% initial fresh mass through needle abscission. To determine needle loss, all dropped needles were collected each day and weighed. Data were subjected to non-linear regression analysis (Sigma Plot 11, Systat Software Inc., Chicago, IL, USA), using the following general logistic equation: a y= x x0 ( ) 1+ e b In the above equation, y represents NRD while x represents light exposure time. The remaining variables a, b, and x0 are constants determined by regression for each relationship. The numerator of the equation, a, is of particular interest because it represents the approximate limit of NRD as exposure time increases. A t-test at 5% signicance was used to test for signicant differences in a between treatments. Constant exposure to LEDs: The second experiment investigated the effect of constant exposure to different sources of light on needle abscission in balsam r branches. The experiment followed a complete randomized design with four replicates, where a single

* Fluorescent lights have many peak wavelengths in the range of 400 -800 nm. ** White LEDs are considered multicolored which uses a combination of red, green, and blue wavelengths to create white light.

branch served as a replicate and each branch was placed in a separate chamber. Each light source (growth chamber uorescent, blue, red, white, or dark) was a treatment. Flasks, with branches, were placed into trays at the bottom of the chamber to collect the needles that had fallen off. Needles were collected with growth chamber lights turned off; the only light was provided by the chamber LED panel to ensure that they were not exposed to any other light during the experiment. For the dark treatment, a lamp with a single 40-watt green incandescent bulb was turned on in the far side of the growth chamber to allow needle collection. After needle collection, branches were returned to their respective treatment. The response variable was NRD (as described above). Data were subjected to an analysis of variance and means separation was performed using the least signicant difference at 5% signicance (SAS v9, SAS Institute Inc, Cary, NC).

Results
Exposure to cool white fluorescent lights or darkness had no signicant effect on NRD in balsam r, with an average NRD of 31.5 days and 35 days, respectively. However, there were significant relationships between NRD and exposure duration to each of red, white, and blue LEDs (Fig. 1). A 48hour exposure to red, white, or blue LEDs increased NRD by approximately 36, 42, and 25 days compared to their respective non-LED exposed treatments. The horizontal asymptote of each relationship represents the limit of effectiveness of each light exposure. A comparison of the horizontal asymptote of each relationship suggests that exposure to red, white, and blue LEDs have a signicantly higher asymptote than uorescent or dark treatments, but there is no signicant difference among the three LED treatments (Fig. 2). A comparison of branch needle loss after 12-, 24-, or 48-hour exposure to each light source is shown in Fig. 3. Continuous exposure to LEDs also had a signicant effect on NRD. The uorescent light and dark treatment each had an NRD of 62 days. However, exposure to red, white, and blue LEDs resulted in a signicantly (P < 0.01) higher NRD of 68, 73, and 76 days, respectively (Fig. 4).

Discussion
Exposure to any LED spectra tested delayed needle abscission, while branches in the dark lost needles on an average 10 to 30 days earlier. The results are consistent with the literature as plants

Effect of LEDs on postharvest balsam r needle retention

15

Fluorescent

Darkness

White

Red

Blue

Fig. 1. Needle retention duration of different exposure times to uorescent lights, darkness, white LEDs, red LEDs, or blue LEDs. A logistic curve is tted to each set of data using exposure duration as the explanatory variable and needle retention duration as the response based on 28 observations.

in the dark will induce senescence faster than those exposed to light (van Lieburg et al., 1990). However, the branches in the dark treatment lost their needles at nearly the same time as the ones in the growth chamber exposed to uorescent lighting. This suggests that there was no preconditioning effect due to short term dark exposure. The general response in NRD due to LED treatments was similar for all spectra tested. The improvement in NRD was minimal after short exposures of 1 to 8 hours, increased sharply after 12 to 24 hours of exposure, and then leveled off. Exposure to uorescent lights or darkness had a similar trend, though the benet to NRD was much lower or nonexistent. Overall, after 48 hour exposure, the white and red LEDs had the greatest effect, delaying abscission for 75 days and 67 days, respectively. Although carbohydrate status was not analyzed in this study, it is possible that carbohydrate synthesis was altered

Fig. 2. Comparison of needle retention duration curve after exposure to uorescent lights, darkness, blue LEDs, red LEDs, or white LEDs (as calculated from each regression in Fig. 1). Numbers in parentheses are the limit of needle retention (in days) for each relationship, based on regression analysis. Letter groupings were calculated from logistic regression coefficient and standard error and indicate a significant difference at = 0.05.

when branches were exposed to LEDs compared to the uorescent light or dark treatment and this may influence abscission. Changes in carbohydrate metabolism are associated with cold acclimation, which similarly alter abscission in balsam r (Beck et al., 2004; MacDonald and Lada, 2008). More specically, in

16

Effect of LEDs on postharvest balsam r needle retention

Fig. 3. Comparison of balsam r branches after exposure to uorescent lights, darkness, blue LEDs, red LEDs, or white LEDs for 12, 24, or 48 hours. Photos were taken 60 days after the start of the experiment. The effectiveness of white LEDs is particularly evident from this photo.

several conifers sucrose and rafnose increased in the winter, strongly correlated with minimum temperatures (Hinesley et al., 1992). Although photosynthesis (Tennessen et al., 1993) and plant growth (Okamoto et al., 1996; Yanagi et al., 1996) are shown to occur under red LED light and may be enhanced with blue LED light, it is not clear that photosynthesis or its resulting carbohydrates played a major role in this study. It should be noted that the cool white light treatment resulted in a NRD similar to dark treatment, even though it provided a much higher quantity of light that could contribute to photosynthesis than any of the LED treatments. Thus any consideration of carbohydrate synthesis should focus on changes in individual sugars instead of overall carbohydrate status. Constant exposure to red, white, or blue LEDs signicantly delayed needle abscission compared to the fluorescent light or dark treatments, but the relative benet was not as high as that observed with short-term exposure. This is largely due to a much higher NRD observed in this experiment. In experiment 1, the typical NRD of branches exposed to uorescent lights was approximately 32 days, but in experiment 2 the same treatment had a NRD of 62 days. It is possible that the difference in NRD is due to differences in harvest time and dormancy status; branches

Fig. 4. Comparison of needle retention in balsam r branches after constant exposure to different light sources. Different letters indicate signicance at 5% signicance as determined using LSD multiple means comparison. Means are calculated from four replicates.

Effect of LEDs on postharvest balsam r needle retention in the experiment 2 were harvested 6 weeks later (at the end of September). Later harvests benet balsam r needle retention and diminish the effect of some abscission mitigating technologies (MacDonald and Lada, 2008; MacDonald et al., 2010a). LEDs have several unique advantage over conventional lighting systems used today in growth chambers and greenhouses. The potential use of LEDs as an eco-friendly postharvest treatment during storage and transport for the Christmas tree industry in Atlantic Canada is promising. The ability to control spectra, their small size, durability, and relatively long lifespan are all traits that growers could easily adapt into their current production practice. However, the biggest advantage is their relatively cool emitting surface and ability to match wavelengths to specic tree photoreceptors to provide optimal balance between plant morphology and metabolism (Bourget, 2008; Massa et al., 2008; Morrow, 2008). LEDs are currently being used in several plant production systems, but are becoming more popular with micropropagation studies because they are more suitable than uorescent lamps (Li et al., 2010). The initial cost of installing custom lights may be high, but could be recovered in a relatively short time as LEDs are very energy efcient. For example, the 12 x 12 inch LED panels used for this experiment used only 13.8 watts of electricity and covered an area of two square feet (LED wholesalers, 2010). However, further study of the LED technology on full size balsam r trees is needed before this technology can actually be implemented on large scale. In conclusion, needle retention was signicantly improved with the red, white, or blue LEDs. Needle abscission was delayed by 118, 127, and 75% after a 48-hour exposure to red, white, or blue LEDs, respectively, compared to uorescent lights. In addition, constant exposures to red and white LEDs delayed abscission by 23 and 18%, respectively compared to uorescent lights. The improvement in needle retention after LED exposure makes LEDs an interesting technology to explore for postharvest Christmas trees.

17

Acknowledgements
We thank the Christmas Tree Council of Nova Scotia for the funding of this project and Aru Thiagarajan for his initial help in the set up of this experiment. We also thank internal reviewers Dr. Peter Havard and Dr. Sanu Jacob for their contributions.

References
Barta, D.J., T.W. Tibbitts, R.J. Bula and R.C. Morrow, 1992. Evaluation of light emitting diode characteristics for a space-based plant irradiation source. Advances Space Res., 12: 141-149. Beck, E.H., R. Heim and J. Hansen, 2004. Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. J. Biosci., 29: 449-59. Bourget, C.M. 2008. An introduction to light-emitting diodes. HortScience, 43: 1944-1946. Bula, R.J., R.C. Morrow, T.W. Tibbitts, D.J. Barta, R.W. Ignatius and T.S. Martin, 1991. Light emitting diodes as a radiation source for plants. HortScience, 26: 203-205.

Chanishvili, S.H. and M.D. Dolidze, 1974. The effect of light intensity on carbohydrate assimilation and distribution in the organic compounds of grapevine leaves. Trudy Inst. Bot., 27: 315-326. Chastagner, G.A. and D.M. Benson, 2000. The Christmas tree: tradition, production, and diseases. Plant Health Progress, DOI:10.1094/PHP2000-1013-01-RV. Chastagner, G.A. and K.L. Riley, 2003. Postharvest quality of noble and Nordmann r Christmas trees. HortScience, 38: 419-421. Christmas Tree Council of Nova Scotia, 2010, <http://www.ctcns.com/ ab_industry.htm. Accessed 05 May 2010>. Curtis, R.W. 1978. Phytochrome involvement in the induction of resistance to dark abscission by malformin. Planta, 141: 311-314. Hinesley, L.E., D.M. Pharr, L.K. Snelling and S.K. Funderburk, 1992. Foliar rafnose and sucrose in four conifer species: relation to seasonal temperature. J. Amer. Soc. Hort. Sci., 117: 852-855. LED wholesalers, 2010. LED wholesalers.com. LED grow light [Online]: http://www.ledwholesalers.com/store/index.php?act=viewProd&pr oductId=370 Accessed 07/11/2010. Li, H., X. Zhigang and C. Tang, 2010. Effect of light-emitting diodes on growth and morphogenesis of upland cotton (Gossypium hirsutum L.) plantlets in vitro. Plant Cell Tiss. Organ Cult., 103: 155-163. MacDonald, M.T. and R.R. Lada, 2008. Cold acclimation can benet only the clones with poor needle retention duration (NRD) in balsam r (Abstr.). HortScience, 43: 1273. MacDonald, M.T., R.R. Lada, M. Dorais, S. Pepin, Y. Desjardins and A.I. Martynenko, 2010a. Ethylene exposure duration affects postharvest needle abscission in balsam r (Abies balsamea L.). HortScience, 46: 260-264. MacDonald, M.T., R.R. Lada, A.I. Martynenko, M. Dorais, S. Pepin and Y. Desjardins, 2010b. Ethylene triggers needle abscission in root-detached balsam r. Trees, 24: 879-886. Massa, G.D., H.H. Kim, R.M. Wheeler and C.A. Mitchell, 2008. Plant productivity in response to LED lighting. HortScience, 43: 19511956. Mitcham-Butler, E.J., L.E. Hinesley and D.M. Pharr, 1988. Effects of harvest date and storage temperature on the postharvest needle retention of Fraser r branches. J. Environ. Hort., 6: 1-4. Morrow, R.C. 2008. LED lighting in horticulture. HortScience, 43: 1947-1950. Okamoto, K., T. Yanagi, S. Takita, M. Tanaka, T. Higuchi, Y. Ushida and H. Watanabe, 1996. Development of plant growth apparatus using blue and red LED as articial light source. Acta Hort., 440: 111-116. Saebo, A., T. Krekling and M. Appelgren, 1995. Light quality affects photosynthesis and leaf anatomy of birch plantlets in vitro. Plant Cell Tissue Organ Culture, 41: 177-185. Senger, H. 1982. The effect of blue light on plants and microorganisms. Phytochem. Photobiol., 35: 911-920. Tennessen, D.J., E.L. Singsaas and T.D. Sharkey, 1993. Light emitting diodes as a light source for photosynthesis research. Photosynthesis Res., 39: 85-92. van Lieburg, M.J., W.G. van Doorn and H. van Gelder, 1990. Prevention of phytochrome-related postharvest loss of quality in ornamentals through red light emitted by diodes. Acta Hort., 272: 347-351. van Meeteren, U. and A. van Gelder, 2000. The role of leaves in photocontrol of ower bud abscission in Hibiscus rosa-senesis L. Nairobi. J. Amer. Soc. Hort. Sci., 125: 31-35. Yanagi, T., K. Okamoto and S. Takita, 1996. Effects of blue, red, and blue/ red lights of two different PPF levels on growth and morphogenesis of lettuce plants. Acta Hort., 440: 117-122.
Received: March, 2011; Revised: October, 2011; Accepted: December, 2011

Journal

Journal of Applied Horticulture, 14(1): 18-24, 2012

Appl

Effect of decit drip-irrigation scheduling regimes with saline water on pepper yield, water productivity and soil salinity under arid conditions of Tunisia
K. Nagaz*, M. M.Masmoudi1 and N. Ben Mechlia1 Institut des Rgions Arides, 4119 Mdenine, Tunisia. 1INAT, 43 avenue Charles Nicolle, 2083 Tunis, Tunisia. *E-mail: Nagaz.Kameleddine@ira.rnrt.tn

Abstract
A two-year study was carried out to assess the effect of different irrigation scheduling regimes with saline water on soil salinity, yield and water productivity of pepper under actual commercial-farming conditions in the arid region of Tunisia. Pepper was grown on a sandy soil and drip-irrigated with water having an ECi of 3.6 dS/m. Four irrigation treatments were based on the use of soil water balance (SWB) to estimate irrigation amounts and timing while the fth consisted of using farmers practices. SWB methods consisted in replacement of cumulated ETc when readily available water is depleted with levels of 100% (FI), 80% (DI-80) and 60% (DI-60). FI was considered as full irrigation while DI-80 and DI-60 were considered as decit irrigation regimes. Regulated decit irrigation regime where 40% reduction is applied only during ripening stage (FI-MDI60) was also used. Farmer method consisted of applying the producer method corresponding to irrigation practices implemented by the local farmers. Results on pepper yield and soil salinity are consistent between the two-year experiments and showed signicant difference between irrigation regimes. Higher soil salinity was maintained over the two seasons, 2008 and 2009, with DI-60 and FM treatments than FI. FI-MDI60 and DI-80 treatments also resulted in low ECe values. Highest yields for both years were obtained under FI (22.3 and 24.4 t/ha) although we didnt nd signicant differences with the regulated decit irrigation treatment (FI-DI60). However, DI-80 and DI-60 treatments caused signicant reductions in pepper yields through a reduction in fruits number/m and average fruit weight in comparison with FI treatment. The FM increased soil salinity and caused signicant reductions in yield with 14 to 43%, 12 to 39% more irrigation water use than FI, FI-MDI60 and DI-80 treatments in 2008 and 2009, respectively. Yields for all irrigation treatments were higher in the second year compared to the rst year. Water productivity (WP) values reected this difference and varied between 2.31 and 5.49 kg/m3. The WP was found to vary signicantly among treatments, where the highest and the lowest values were observed for DI-60 treatment and FM, respectively. FI treatment provided signicant advantage on yield and water productivity, compared to FM in pepper production under experimental conditions. For water-saving purposes, the FI irrigation scheduling is recommended for drip irrigated pepper grown under eld conditions and can be used by farmers to optimize the use of saline water and to control soil salinity. In case of limited water supply, adopting decit irrigation strategies (FI-DI60 and DI-80) could be an alternative for irrigation scheduling of pepper crop under the arid Mediterranean conditions of Tunisia. Key words: Arid, salinity, drip irrigation, irrigation scheduling, decit irrigation, pepper, yield, water productivity

Introduction
Water is becoming increasingly scarce, creating droughts which are becoming still more serious due to changing climate conditions, especially in the southern Mediterranean region. Restricted supply of good quality water is the major limiting factor for crop production in arid regions of Tunisia. Presently, there is an increasing tendency to use saline water in this region to intensify agriculture. Irrigation of a wide range of vegetable crops such as potatoes, lettuces, carrots and peppers is expanding around shallow wells having a TDS more than 1.7 g/L. In the absence of sufficient rainfall for natural leaching, irrigated farming is exposed to accumulation of salts in the soils. Several studies have indicated that when saline water is used for irrigation, due attention should be given to minimize root zone salinity (Gideon et al., 2002; Katerji et al., 2004). Others have indicated the need to select appropriate irrigation systems and practices that will supply just a sufcient quantity of water to the root zone to meet the evaporative demand and minimize salt accumulation inside (Bresler et al., 1982; Munns, 2002).

Efficient use of irrigation water is becoming increasingly important, and alternative water application method such as drip, may contribute to the best use of water for agriculture and improving irrigation efciency. With the drip irrigation systems, water and nutrients can be applied directly to the crop at the root level, having positive effects on yield and water savings (Phene and Howell, 1984). Ayers et al. (1986) and Saggu and Kaushal (1991) showed that saline water can be efciently used through drip irrigation even on saline soils. Moreover, it results in considerable saving in irrigation water (Yohannes and Tadesse, 1998) thus reducing the risks of salinization. However, complementary approaches are still needed to increase WUE. In areas of recurrent water scarcity, decit irrigation (DI) is a common practice to mitigate yield reductions (Kirda et al., 1999). DI involves irrigating the entire root zone with less than full evapotranspiration (ETc) throughout the season. Worldwide, successful attempts have been documented regarding the use of decit irrigation method to improve irrigation WUE in various crop species (Grant et al., 2004; Romero et al., 2004; Cifre et

Effect of decit drip-irrigation scheduling regimes with saline water on pepper al., 2005; Tognetti et al., 2005; Dorji et al., 2005; Wakrim et al., 2005). The decline in water availability for irrigation and the positive results obtained in some fruit tree crops have renewed the interest in developing information on decit irrigation for a variety of crops (Dorji et al., 2005; Fereres and Soriano, 2007). Pepper (Capsicum annuum L.) for fresh market production is rather common in the arid areas of Tunisia. This crop, classied as a sensitive crop to water stress (Doorenbos and Kassam, 1986), is grown during spring-autumn period in individual plantings usually not exceeding 1-2 ha and irrigated with water from shallow wells. Such sensitivity has been documented in several reports (Antony and Singandhupe, 2004; Sezen et al., 2006). For high yields, an adequate water supply and relatively moist soils are required during the growing season. A signicant yield reduction was reported by limiting the irrigation amount during different growing periods such as vegetative, owering or fruit settings (Doorenbos and Kassam, 1986). Della Costa and Gianquinto (2002) reported that continuous decit irrigation signicantly reduced total fresh weight of fruit, and the highest marketable yield was found at irrigation of 120% ET; lowest at 40% ET, and marketable yield did not differ among 60, 80 and 100% ET. Antony and Singandhupe (2004) reported that pepper yield was less at lower levels of irrigation. Dorji et al. (2005) compared traditional drip system irrigation to decit irrigation (DI) for hot pepper and found that water savings with DI were about 50% of traditional drip irrigation. Studies on the water requirements of horticultural crops in arid regions of Tunisia are limited and irrigation is mainly scheduled according to farmers experience, despite the water scarcity. Because pepper crop is of high economic value, the irrigation management strategy seeks maximum yield by supplying all requirement of the crop. However, irrigation is typically applied on a routine basis without scheduling and supply often exceeds crop requirements. To gain information on water requirements of pepper crop, eld investigations were initiated in 2008 with the objective to determine irrigation water requirements of pepper crop and to make quantitative assessments of both soil salinity and yield response to water supply in relation to decit irrigation strategies with saline water in order to derive an irrigation strategy that save water in irrigated pepper, reduce salt input and improve crop water productivity under the arid conditions of Tunisia. With the expectation to enable growers to incorporate more appropriate irrigation scheduling and decit irrigation methods in their usual production practices, all eld work was conducted with farmers participation.

19

during the growing seasons (May through October) was 29.5 and 44.5 mm, respectively, which was lower than the long-term mean rainfall of 54.5 mm (Fig. 1). Most of the rainfall occurred during May, September and October. The monthly reference evapotranspiration (ETo-PM) was similar, though with slightly higher values for the long-term ETo-PM, with a total of 938 mm as compared to 932 and 898 mm, the ETo-PM during the period under experiment for 2008 and 2009. Maximum ETo-PM occurred during July-August (Fig. 1). The soil of the experimental area was sandy with 87.9% sand, 8.9% silt and 3.9% clay. Average values in the 80 cm topsoil of eld capacity (0.33 bar, pF 2.5) and permanent wilting point (15 bar, pF4.2) were, respectively, 12.0 and 3.6% and organic matter concentration was 7.6 g/kg. The soil bulk density for 0-0.8 m depth was 1.49 g/cm3. The total soil available water calculated between eld capacity and wilting point for an assumed pepper root extracting depth of 0.80 m, was 100.5 mm. The electrical conductivity (ECe) values measured before transplanting of pepper seedlings were 3.1 and 2.7 dS/m for rst and second year, respectively. Crop management and experimental design: Same amounts of fertilizers were supplied during both the cropping period. Before transplanting of pepper seedlings, soil was spread with 9.5 t/ha of organic manure. Nutrient supply followed local practices consisting of giving N in the form of ammonium nitrate, P2O5 and K2O at rates of 200, 150 and 150 kg/ha, respectively. The P2O5 and K2O fertilizers were applied as basal dose before transplanting. Nitrogen was divided and delivered with the irrigation water in all treatments during early vegetative growth. All treatment plots received the same amount of fertilizer. Plants of Capsicum annuum (cv. Baklouti), a variety widely used in the region, were gently transplanted into the blocks on 1st May, 2008 and 2nd May, 2009, respectively on the rst and second year of the study. The plants were grown 70 cm apart among the ve rows in each plot with 40 cm spacing between each row, in a randomized complete block design with four replicates and ve irrigation treatments. The same experimental area was used for both years and was divided into four blocks with ve elementary plots per block. Each elementary plot consisted of ve rows. Individual plot size was 48 m2 (12 x 4 m). All plots were drip irrigated with water from a well having an ECi of 3.6 dS/m. Chemical analysis of irrigation water is given in Table 1. Each dripper had a 4 L/h ow rate. Water for each block passed through a water meter, gate valve, before passing through laterals placed in every pepper row. A control mini-valve in the lateral permited use or non-use of the dripper line. The experiments consisted of ve distinct irrigation treatments: The FI treatment considered as full irrigation consists of giving 100% ETc when readily available water in the root zone is depleted. Two additional treatments were irrigated at the same frequency as treatment FI but irrigation amount covered 60 and 80% of cumulated ETc (DI-60 and DI-80). These treatments were identied as continuous decit irrigation treatments. In the fourth treatment (FI-MDI60), considered as regulated decit irrigation regime, water was applied as FI treatment during the transplanting-mid-season period and restricted to 60% of ETc afterwards, until harvest. A fth irrigation treatment consisted of

Materials and methods

Experimental site and climate: The eld experiment was carried out during the growing season of 2008 and 2009, between May and October, in a commercial farm located in the Southern East of Tunisia (3322 N, 906 E; altitude 45 m) in the region of Mdenine. Climate is typically Mediterranean with dry and hot summers and precipitations irregularly distributed throughout the year. Long-term mean monthly climatic data (1979-2002) and climatic data relative to the growing seasons of the period 2008 and 2009 are presented in Fig. 1. Analysis of the climatic data indicated that in 2008 and 2009 growing season temperatures were similar to the typical of long-term means. Rainfall received

20

Effect of decit drip-irrigation scheduling regimes with saline water on pepper

Fig. 1. Long-term mean monthly and growing season climatic data of the experimental area.

applying the farmers method (FM) corresponding to traditional irrigation practices adopted by local farmers where xed amount of water (30 mm) was supplied to the crop every 7 days from transplanting till harvest. The crop evapotranspiration (ETc) was estimated for daily time step by using reference evapotranspiration (ETo) combined with a pepper crop coefcient (Kc) using the dual crop coefcient approach. ETo was estimated using daily climatic data collected from the meteorological station, located at Mdenine, Tunisia and the FAO-56 Penman-Monteith method (ETo-PM) (Allen et al., 1998). The Penman-Monteith method considers hypothetic grass reference crop with a crop height of 0.12 m, a xed surface resistance of 70 s.m-1 and an albedo of 0.23. For irrigation scheduling, the method used was the water balance developed according to the methodology formulated by Allen et al. (1998) and implemented in an Excel spreadsheet program. The program estimates the day when the target soil water depletion (readily available water, RAW) for the treatment FI would be reached and the amount of irrigation water needed to replenish the soil prole to eld capacity. The program calculates, on daily basis, the soil water depletion using the soil water balance and estimates the next irrigation date considering a depletion limit of 40% of total available water in the root zone (TAW). Soil depth of the effective root zone is automatically increased linearly with pepper crop coefcient from a minimum of 0.20 m at transplanting to a maximum of 0.80 m.
Table 1. Chemical composition of irrigation waters (meq/L) ECi Ca2+ (dS/m) +Mg2+ 3.6 25.60 Na+ 9.45 K+ 0.95 Cl8.50 CO32- SARiw +HCO323.00 4.50 2.64 SO42-

Measurements and water-use efciency: Sections of all plots were harvested to determine fresh fruit yield, fruit number and weight each year. In both years, the area of land harvested was 30 m2 per plot depending on the physiological maturity of plants. Occurrence of the harvesting time was recorded as number of days after transplanting (DAT) accordingly. The rst harvest was made on DAT 107, the second harvest was on DAT 133 and nal picking was made on DAT 170 in 2008; and the corresponding harvests for the second year (2009) were on DAT 112, DAT 136 and DAT 170, respectively. The total fruit from each treatment was weighted to determine fresh fruit yield (t/ha). Fruit number was determined by dividing total number of fruits by area of land harvested for each treatment (fruit number/ha). Fresh fruit sub-samples from each treatment were weighted to determine average fruit weight (g/fruit). Water productivity (WP) is dened as the pepper fresh fruit yield obtained per unit of irrigation water applied. The WP was calculated as follows: WP (kg/m3) = Yield (kg/ha) / total irrigation water applied (m3/ha) from transplanting to harvest; an irrigation of 100.5 mm applied before transplanting was not included in the total. Soil samples were collected after harvest and analyzed for ECe. The soil was sampled at every 20 cm to a depth of 80 cm, at four sites perpendicular to the drip line at distances of 0, 10, 20 and 30 cm from the line, and at three sites between the emitters (0, 10 and 20 cm from the emitter). Conceptually, these should be areas representing the range of salt accumulations (Bresler, 1975; Singh et al., 1977). Statistical analysis: Experiments were designed as randomized complete blocks, with each replicate representing a separate block.

Effect of decit drip-irrigation scheduling regimes with saline water on pepper Treatment effects on pepper yields and components, WP and soil salinity were analyzed using analysis of variance (ANOVA) procedure of STATGRAPHICS Plus 5.1 (www.statgraphics.com). Least signicant difference (LSD) test at P 0.05 was used to nd any signicant difference between treatment means.

21

Results and discussion

Evapotranspiration estimates and soil water balance: Fig. 2 illustrates the course of daily ETc relative to ETo for both years during the growing periods of pepper crop. During the rst 35 days after transplantation, high ETo values resulted in high ETc despite the low crop cover. Frequent wetting of the soil surface by irrigation or precipitation increased soil evaporation, controlled mainly by soil hydraulic properties and solar radiation. This period is characterized by mean values of ETc of about 1.7 and 1.2 mm/day, respectively, for the rst and second year. As the crop canopy grew, ETc increased and reached its highest mean value at mid-season stage (5.4 and 5.5 mm/ day). The mean ETc values at the late stage were about 5.6 and 5.8 mm/day for 2008 and 2009 respectively. The high ETc values during the late stage were mainly attributed to the soil evaporation induced by the frequency of irrigation or precipitation and to the relatively high evaporative demand. The spreadsheet program uses water balance equation and gives estimations of the date and amounts of irrigation based on cumulative soil water depletion. Fig. 3 illustrates soil water depletion, estimated by the program, under FI treatment during the cropping period of pepper for two years. This gure also illustrates the effect of an increasing root zone on the readily available water. The rate of root zone depletion at a particular time in the season is given by the net irrigation requirement for that period. Each time the irrigation water is applied, the root zone is replenished to eld capacity. Because irrigation is not applied in the program until the soil water depletion at the end of the previous day is greater than or equal to the readily available water, occasionally plants could be subject to a slight stress on the day prior to irrigation. Soil salinity: The initial and nal average ECe values in the 0-80 cm soil layer under different irrigation treatments are presented in Fig. 4. Initial soil salinity values determined at transplanting were, respectively, 3.1 and 2.7 dS/m in the rst and second year. The results show that during the two years, an increase in ECe values measured in the root zone (0-80 cm) at harvest was observed

Fig. 2. Estimated daily ETc for pepper crop during the cropping seasons.

under all irrigation treatments compared to initial soil salinity. The ECe for the treatments FI and FI-MDI60 increased from 3.1 and 2.7 at transplantation in May to approximately 4.1 and 3.7 dS/m at harvest, respectively, for 2008 and 2009. However, the ECe was higher at harvest than the initial ECe for DI-80, DI-60 and FM treatments as compared to FI and FI-MDI60 treatments. The higher soil salinity obtained for all irrigation treatments at harvest during the two years may be attributed to the high evaporative demand during the cropping period and since rainfall received during that period was 29.5 and 44.5 mm, respectively and water supply was provided mostly through irrigation, little leaching of the soil was expected. The highest ECe values occurred during

Fig. 3. Estimated daily soil water depletion under FI irrigation treatment during the cropping seasons of pepper (2008-2009)

22

Effect of decit drip-irrigation scheduling regimes with saline water on pepper

Fig. 4. Soil salinity (ECe, dS/m) under different irrigation treatments.

Fig. 5. Fresh fruit yield under different irrigation treatments.

the rst year, and the lowest was attained during the second year. The low values of ECe in second year were due to the relatively low initial soil salinity and the leaching of soluble salts with the relatively higher amount of rainfall received (Fig. 1). The total rainfall during pepper growing season of second year was higher than the rst year (Fig. 1). Data shows that ECe decreased in full irrigation treatment (FI). FI-MDI60 and DI-80 irrigation treatments resulted in low ECe values. The ECe values were not signicantly different between FI, DI-80 and FI-MDI60 treatments. However, higher soil salinity was observed in case of DI-60 decit irrigation treatment and FM than FI treatment. The reason for the higher soil salinity obtained for decit irrigation treatment (DI-60) is attributed to absence of substantial leaching under decit irrigation conditions. Geerts et al. (2008), Kaman et al. (2006) and Schoups et al. (2005) reported that one consequence of reducing irrigation water by decit irrigation is the greater risk of increased soil salinity due to reduced leaching. With the FM treatment, irrigation is typically applied without scheduling and application of irrigation water frequently exceeds crop requirements. Over irrigation helps to leach salt below the root zone during the rst few periods of cultivation, but it carries the danger of a rapid soil salinization because of increased salt input. Thus, the higher ECe values obtained under FM treatment may be attributed to the fact that more irrigation water under conditions of high evaporative demand would result in higher direct evaporation rates leading to an increase in salt accumulation in the soil. Crop yield: Pepper yields data are presented in Fig. 5. The data shows that pepper yield over the two years of the study were affected by irrigation treatments. Fresh fruit yields ranged from 17.97 to 24.40 t/ha in both years. Yields were highest in the second year because of the low soil salinity and the relatively higher amount of rainfall received (44.5 mm). The highest pepper fresh fruit yield was obtained under the FI irrigation treatment. FIMDI60 where water restriction is applied only during the ripening stage also provided higher fruit yield and was not signicantly different with FI, similar to what was found by Gonzlez-Dugo et al. (2007). Yield obtained under FM treatment was 21 and 19% lower, respectively, in 2008 and 2009 and signicantly different (P<0.05) than that obtained with FI treatment. Although 6.2 and 7.1% yield reduction was evident under DI-80 in 2008 and 2009, it was not statistically different than yield of the FI treatment,

respectively. However, a signicant reduction in yields occurred with the DI-60 as compared to FI treatment (Fig. 5). Della Costa and Gianquinto (2002) and Katerji et al. (1993) reported that continuous water stress signicantly reduced fresh fruit yield. Statistically signicant differences (P<0.05) were found between the DI-80 and DI-60 treatments for both years. Among the DI-60 and FM, fresh fruit yield of FM was the lowest but the difference was not signicant (P<0.05). The difference between the FM and other treatments (DI-80 and FI-MDI60) were signicant (P<0.05). The inuence of irrigation treatment on the fruit number and weight was highest for treatment FI and was followed by FIMDI60 and DI-80 treatments in both years (Table 2). Differences between FI, FI-MDI60 and DI-80 treatments were not signicantly different (P<0.05). Statistically signicant differences (P<0.05) were found between FI treatment and DI-60 and FM treatments for each year. There was no signicant difference between DI-60 and FM treatment and DI-80 and DI-60 treatment in fruit weight and number during both years. Fernandez et al. (2005) and Dorji et al. (2005) reported that water decit affect fruit number and weight. Pepper is among the most susceptible horticultural plants to drought stress (Alvino et al., 1994; Dimitrov and Ovtcharrow, 1995). The water decit during the period between owering and fruit development reduced nal fruit production (Jaimez et al., 2000; Fernandez et al., 2005 and Dorji et al., 2005). Note that the decit irrigation treatment (DI-60) and producer method (FM) resulted in higher salinity in the rooting zone than the DI-80, FI and FI-MDI60 treatments (Fig. 4). The higher salinity associated with decit irrigation DI-60 and FM treatments were sufcient to cause reduction in pepper yield through a reduction in fruits number and weight (Table 2). The yield is greatly dependant on timing, amount and frequency of irrigation applied. Lower yields obtained under FM treatment may be attributed to the fact that the farmer applies water to the crop regardless of the effective plant needs. This seems to relate irrigation occurrences to days after transplanting rather than to progress of crop growth stages. The corresponding irrigation applications are often characterized by periods of over- and under-irrigation. Raes et al. (2002) reported that excess watering in saline conditions may cause loss of valuable nutrients out of the root zone and soil salinization, especially during crop

Effect of decit drip-irrigation scheduling regimes with saline water on pepper sensitive periods, which results in limited growth and reduction in crop yield. Irrigation scheduling based on crop water requirements and soil characteristics allows for applying irrigation water when needed during the growing season. However, its application is only possible when water supply and irrigation amounts can be managed independently by farmers (Smith, 1985). In areas where pepper is irrigated with well waters, accurate scheduling is manageable. This is precisely the case of our area; therefore there is a high chance to optimize water supply to crops. Water supply and productivity: Amounts of irrigation water and total water supply for each irrigation treatment during the two years are presented in Table 3. Irrigation water applied before transplanting of pepper (100.5 mm) each year is not included in the total. For all treatments, total water supply ranged from about 420 to 780 mm. With the producer method (FM) more irrigation water was used than the FI and decit irrigation treatments. Surplus was 94 to 356 mm in 2008 and 77 to 339 mm, in 2009. Rainfall was 29.5 mm in the rst year and 44.5 mm in the second year. For FI treatment, irrigation amounts of the both years were quite similar with 656 mm in 2008 and 654 mm in 2009. Using the FI-MDI60 strategy, 77 and 53 mm of water was saved in the rst and second year, respectively. Similarly, the water savings achieved with DI-80 and DI-60 treatments were 131 and 262 mm compared to the FI treatment. TWP and IWP values reported in this study were similar to those reported for pepper by Genolan et al. (2006) and Dadelen et al. (2004) and were signicantly inuenced by the irrigation treatments (Table 3). There is also a variation in WP values between years. For all irrigation treatments, yield was higher in the second year compared to the rst year. Values of water productivity of irrigation (IWP) reflect this difference; they varied typically around 2.4-4.84 and 2.7-5.49 kg/m3 in the rst and second year, respectively. TWP values ranged from 2.31 kg/m3 in FM to 4.93 kg/m3 in the DI-60. IWP values varied from a minimum of 2.4 kg/m3 in FM to a maximum of 5.49 in kg/m3 in the DI-60 treatment in the experimental years. For both the years, WP values of FM and full irrigation (FI) treatments were considerably lower than those of the decit treatments. The WP with FI treatment was not significantly different from those obtained with FI-MDI60 treatment but statistically different from those obtained with DI-80, DI-60 and FM treatments. These three last treatments show a statistical difference between them. The low irrigation water productivity for the producer method (FM) during the two experiments can
Table 2. Yield components under different irrigation treatments Treatment 2008 2009 Fruits number Average Fruits number Average (1000/ha) fruit weight (1000/ha) fruit weight (g/fruit) (g/fruit) FI 1046 21.297 1061 23.011 DI-80 1006 20.278 1012 22.201 DI-60 991 19.219 1004 21.102 FI-MDI60 1022 21.039 1026 23.005 FM 983 18.290 998 19.997 LSD (5%) 54 1.114 50 1.107

23

be attributed to reduced yields and also to higher irrigation water use. In this study, our results demonstrate that the effects of irrigation treatments are signicantly important in order to obtain higher yields of eld grown pepper under the Mediterranean climatic conditions in Tunisia. Irrigation treatments had signicant effect on soil salinity, pepper yield and its components parameters. Full irrigation (FI) and decit irrigation treatments (FI-MDI60 and DI80) decreased the soil salinity. Higher soil salinity developed in the root zone with DI-60 decit irrigation and farmer method (FM). Fresh fruit yields of decit irrigated treatments (DI-60 and DI-80) were signicantly lower than those in full irrigation treatment (FI) which had the lowest soil salinity. Treatment FI-MDI60 also gave good yields. Moreover, FI and FI-MDI60 treatments resulted in better yield components parameters such as the number of fruits and fruit weight as compared to other treatments. Note that the decit irrigation treatments gave lower yields and resulted in higher salinity in the rooting zone than the full irrigation (FI). The xed amount approach used by the farmer was the least efcient and caused higher salinity in the rooting zone. This method gave the lowest fresh fruit yields with 14 to 43%, 12 to 39% more irrigation water applied than FI, FI-MDI60 and DI-80 treatments, respectively, in 2008 and 2009. The data show that factors such as fruit number and weight are signicant components of pepper yield. The higher salinity associated with the farmers method and decit irrigation treatments caused reduction in fresh fruit yield and yield components. The water productivity for fresh fruit yield was signicantly affected by irrigation treatments. The lowest values occurred under the FM treatment, while the highest values were obtained under deficit irrigation treatment DI-60. High efficiencies observed for the most severe restricted regime (DI-60) is therefore counterbalanced by reduced yield and quality. The relatively high yields and water productivity values obtained under DI-80 and FI-MDI60 treatments indicate the high potential of the pepper crop to valorize irrigation waters of poor quality under mild water decit conditions. FI-MDI60 and DI-80 saved water by 8-20%, reduced soil salinization and improved irrigation water
Table 3. Water supply (mm) and productivity (WP, kg/m3) for different irrigation treatments in both years Treatment Irrigation* (mm) FI DI-80 DI-60 FI-MDI60 FM LSD (5%) FI DI-80 DI-60 FI-MDI60 FM LSD (5%) 656 525 394 579 750 654 523 392 601 731 Rainfall (mm) 2008 29.5 29.5 29.5 29.5 29.5 2009 44.5 44.5 44.5 44.5 44.5 698.5 567.5 436.5 645.5 775.5 3.73 4.31 5.49 3.93 2.70 0.34 3.49 3.97 4.93 3.66 2.55 0.28 685.5 554.5 423.5 608.5 779.5 3.40 3.89 4.84 3.71 2.40 0.36 3.25 3.68 4.50 3.53 2.31 0.34 I+R (mm) IWP (kg/m3) TWP (kg/m3)

* an irrigation of 100.5 mm supplied just before transplanting is not included in these totals

24

Effect of decit drip-irrigation scheduling regimes with saline water on pepper

FAO Report, 2002. Decit Irrigation Practices. FAO, Rome, p. 102. Fisher, R.A., 1980. Inuence of water stress on crop yield in semiarid regions. In: Crop Water Requirements. Conf., 11-4 September 1984, Paris, INRA, Paris, N.C. Turner and P.J. Kramer (Eds.), pp. 221-234. Hutton, R. 2000. Farmers newsletter, 2000: improving the water use efciency of citrus at Yanco Agricultural Institute. Hort., 184: 4749. Jones, H.G. 2004. Irrigation Scheduling: advantages and pitfalls of plant-based methods. J. Exp. Bot., 55: 2427-2436. Kang, S., H. Xiaotao, I. Goodwin and P. Jerie, 2002. Soil water distribution, water use, and yield response to partial rootzone drying under a shallow groundwater table condition in a pear orchard. Sci. Hort., 92: 277-291. Katerji, N., J.W. van Hoorn, A. Hamdy and M. Mastrorilli, 2003. Salinity effect on crop development and yield analysis of salt tolerance according to several classication methods. Agric. Water Manage., 62: 37-66. Kirda, C., M. Cetin, Y. Dasgan, S. Topcu, H. Kaman, B. Ekici, M.R. Derici and A.I. Ozguven, 2004. Yield response of greenhouse grown tomato to partial root drying and conventional decit irrigation. Agric. Water Manage., 69: 191-201. Kirnak, H. and M.N. Demirtas, 2006. Effects of different irrigation regimes and mulches on yield and macronutrition levels of dripirrigated cucumber under open eld conditions. J. Plant Nutr., 29: 1675-1690. Oster, J.D., 1994. Irrigation with poor quality water-review article. Agric. Water Manage., 25: 271-297. Shalhevet, J., 1994. Using water of marginal quality for crop production: major issues. Agric. Water Manage., 25: 233-269. Shani, U. and L.M. Dudley, 2001. Field studies of crop response to water and salt stress. Soil Sci. Soc. Am. J., 65: 1522-1528. Smittle, D.A., W.L. Dickens and J.R. Stansell, 1994. Irrigation regimes affect yield and water use by bell pepper. J. Am. Soc. Hortic. Sci., 119(5): 936-939. Stanghellini, C., F.L.K. Kempkes and P. Knies, 2003. Enhancing environmental quality in agricultural systems. Acta Hort., 609: 277-283. van Hooijdonk, B.M., K. Dorji and M.H. Behboudian, 2004. Responses of Pacic Rose apple to partial rootzone drying and to decit irrigation. Eur. J. Hortic. Sci., 69: 104-110.
Received: October, 2011; Revised: November, 2011; Accepted: January, 2012

productivity. Although DI-80 and FI-MDI60 treatments reduced fruit number and weight, fresh fruit yield was maintained, when compared to well-irrigated treatment Fl and had higher fresh fruit yield than the farmer treatment (FM). In conclusion, FI treatment is recommended for drip irrigated pepper grown under eld conditions and can be used by farmers to schedule irrigation of pepper in order to obtain higher yield in the Mediterranean region of Tunisia. The results of this study suggest that the DI-80 and FI-MDI60 practices can be viable and advantageous option next to FI to reduce soil salinization and prevent crop yield reduction when and if there is water shortage. Decit irrigation (DI) can only be successful if measures are taken to avoid salinization since leaching of salts from the root zone is lower under DI than under full irrigation (FI). The decit irrigation presents a potential to improve the water productivity and the control of soil salinization when it can benet from the leaching capacity of rains. Future investigations should focus on this issue and evaluate the efciency of the small amounts of rain that occur in spring-fall for natural leaching.

Acknowledgements
We are grateful to Mohsen Chandoul and Hedi Hajjaji for technical assistance, to farmer for his contribution in this study.

References
Arzani, K., D. Arzani and G.S. Wood, 2000. Inuence of rst season application of paclobutrazol, root-pruning and regulated decit irrigation on second season flowering and fruiting of mature Sundrop apricot trees. Acta Hort., 516: 75-82. evik, B., K. Abak, N. Sari, C. Kirda and F. Topalolu, 1996. Effects of different irrigation levels on yield and quantity of some vegetable irrigated with drip irrigation in Harran Plain. Project No: 16, Agric. Faculty General Pub. No. 169, Adana. Delne, S., F. Loreto and A. Alvino, 2001. Drought-stress effects on physiology, growth and biomass production of rainfed and irrigated bell pepper plants in the Mediterranean region. J. Am. Soc. Hortic. Sci., 126(3): 297-304. English, M.J. and S.N. Raja, 1996. Perspective of decit irrigation. Agric. Water Manage., 32: 1-14.

Journal

Journal of Applied Horticulture, 14(1): 25-28, 2012

Appl

Observations on leaf morphology of male and female Actinidia chinensis plants

W. Liu, M. Yang and H. Liang* College of Life Sciences, Zhongkai University of Agriculture and Engineering, Guangzhou, China. *E-mail: lhoce@yahoo.com.cn

Abstract
Differences of leaf morphology between male and female plants of Actinidia chinensis were observed by means of microscopic and scanning electron microscope (SEM) observations. The experimental results showed that ratios of guard cell length to width were signicantly different between male and female plants, which were greater than 3 in male plants and lower than 3 in female plants. Leaf shapes and petiole appearance were slightly different among different cultivars, however, the special parameter related to gender could not be found. Male seedlings and female seedlings germinated from seeds in the same fruit could be identied according to ratio of guard cell length to width. It is suggested that ratio of guard cell length to width may be used as a good marker to distinguish male plants from female plants in A. chinensis. Key words: Actinidia chinensis, gender identication, leaf, morphology

Introduction
Kiwifruit plants are functionally dioecious plants and belong to Actinidia (Yang et al., 2010). There is gender diversity in Actinidia and the sex differentiation of kiwifruit plants is in primitive evolution, thus Actinidia can be used as the model plants for studies on sex origin, evolution, determination, and reproduction biology (Yang et al., 2009). Bugala in the 1950s, identied male and female Populus tremula by difference in leaf color (Li et al., 2006). Gong (1995) studied leaf shape, crown layer, bark and lenticel of the seedlings and mature plants of Populus talassica in summer and winter, and suggested the morphological parameters for gender identication. Lian et al. (2000) measured the leaves of Hippophae rhamnoides and found the female plants had usually larger ratio of leaf length to width than the male plants. To search for easy and rapid morphological markers for identifying gender of kiwifruit seedlings in their early developmental stage, comparisons of leaf morphology between male and female Actinidia chinensis were carried out in this study. The experimental results would be helpful in direct use of kiwifruit seedlings in farming production, selection of male or female plants in hybrid seedling population and cultivation of female vigorous F1 plants.

shoots at different positions in each plant were picked up, photographed and determined. After observation on their leaf vein and leaf shape, the leaves were put in FAA solution (70% ethanol + 5% acetic acid + 2% formalin + 23% H2O) for 24 h, and then transferred to 70% ethanol solution for long term storage. The xed leaves were used to make freehand section, and the sections were scanned and photographed under Leica DM4000B microscope system. SEM observation: The leaf samples were treated in FAA solution according to Yang (2010) for 24 h, and then transferred to 70% ethanol solution for long term storage. After dehydration in ethanol series (80%, 90%, 100%, for 10 min in each ethanol solution), the leaf samples were put in tertiary butanol 3 times (5 min each) and dried completely in a freeze dehydrator. The dry leaves were put on ion sputtering coator in which Pt was used as the ion source, and then observed under JSM-6360LV scanning electron microscope.

Results
Observations on leaf appearance of A. chinensis: One year old branch stems of A. chinensis Planch. var. chinensis were greygreen and the two year old branch stems were grey-brown, on which there was little or no hair. Yellow-brown lenticels protruded on the branch stems. Leaves were bifacial and the veins protruded on the lower surface (Fig. 1A-D). The blades were oval, round or round-fan shaped and the blade bases were heart-shaped and symmetrical. Terminal leaf margin of the blades was curved, obtuse-shaped or concave. The upper leaf surfaces were darkgreen and smooth. The lower leaf surfaces were grey-green and hairy with pale-green hairy veins. The petioles were pale-violet or pale-green, without hair on petiole surface. One year old branch stems of A. chinensis Planch var. deliciosa were green with grey-brown hairs and a few protruded lenticels on them, and the two year old branch stems were red-brown on

Materials and methods

Plant materials: Four cultivars of Actinidia chinensis Planch var. chinensis, viz., Heping Hongyang , Heping Hongyang , Fengxion No. 1 and Wuzhi No. 3 , and 4 cultivars of A. chinensis Planch var. deliciosa, Bangzen No. 1 , Heping No. 1 , Zhaoxia No. 3 and Miliang No. 1 , grown in the germplasm collection of Fruit Research Institute of Heping County (Guangdong, China), were used in this study. Six plants of each cultivar were randomly chosen for leaf samples. The mature green leaves were used for morphological observation. Microscopic observation: Mature green leaves of vigorous

26

Observations on leaf morphology of male and female Actinidia chinensis plants

Table 1. Morphological parameter of kiwifruit leaves (mm) Cultivar Bangzen No. 1 Zhaoxia No. 3 Fengxion No. 1 Heping No. 1 Miliang No. 1 Wuzhi No. 3 Petiole length Petiole diameter Leaf length Leaf width

which there was no hair but a few pale oval-shaped lenticels on them. The leaves were bifacial leaves and the veins protruded on the lower surface (Fig. 1 E-H). The blades were round, oval or round-fan shaped, and the blade bases were shallow-heart shaped and symmetrical. The leaf margins showed small protruded spikes. The upper leaf surfaces were dark-green with a few yellow-brown villi. The lower leaf surfaces were pale-green with crowded yellow epidermal hairs. The leaf veins were yellowgreen or pale-green, with short brown villi on them. Size and shape of the leaves of A. chinensis showed some difference among different cultivars (Table 1). In cultivars of var. chinensis, the relative petiole length of the male plants was

90.83.22 5.410.07 164.64.08 186.22.52 87.25.93 2.830.08 143.42.36 146.85.45 66.43.75 2.790.04 79.82.71 107.62.11 54.85.12 3.710.03 143.45.38 163.66.49 70.26.84 3.510.12 125.24.43 137.64.37 79.08.21 4.560.10 131.64.01 135.44.49

Heping Hongyang 47.61.08 2.090.17 60.32.25 66.23.09

Heping Hongyang 75.95.81 3.940.18 128.67.84 136.06.59

Fig. 1. Leaves of A. chinensis. A. Heping Hongyang , B. Heping Hongyang , C. Fengxion D. Wuzhi No. 3 , E. Bangzen No. 1 , F. Heping No. 1 , G. Zhaoxia No. 3 , H. Miliang No. 1 , a. upper leaf surface, b. lower leaf surface, 1. side vein, 2. main vein, 3. petiole

Observations on leaf morphology of male and female Actinidia chinensis plants

27

Fig. 2. SEM photographs of epidermis of A. chinensis leaves. C-D. upper epidermal hair, G-H. stoma in lower epidermis, I-J. lower epidermal hair

longer than the female plants, while in cultivars of var. deliciosa, the relative petiole length of the male plants was shorter than the female plants. In 8 cultivars studied, ratio of petiole length to petiole diameter of the male plants was more than the matched female cultivars (Bangzen No. 1 > Heping No. 1 , Zhaoxia No. 3> Miliang No. 1 , Fengxion No. 1> Wuzhi No. 3 , and Heping Hongyang > Heping Hongyang ). The petioles of the male plants were relative long and thin. However, since there was variation and overlapping among cultivars or combinations, parameters of leaf shape and petiole appearance could not be used as maker to distinguish male plants from female plants. Microscopic observation on A. chinensis: Sections of leaf blades from 8 cultivars of A. chinensis under microscope showed upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis. The upper epidermis was of one layer of cells

with thickness of 14-22 m. The palisade parenchyma was one to two layers of cells, arranged closely. The spongy parenchyma arranged irregularly. The lower epidermis consisted of 1-5 layers of loosely arranged cells. Leaf veins interspersed among spongy parenchyma and protruded outside. Anatomically, there was little difference between var. chinensis and var. deliciosa, and also little difference between the male plants and the female plants. SEM observations on A. chinensis: SEM observations on A. chinensis showed that there were few and scattered trichomes, seldom branched on the upper leaf surfaces (Fig. 2C-D). The lower leaf surface of var. chinensis spread thick trichomes and villi. Branches of a trichome were 3-19 (Fig. 2I-L). The SEM observations showed little difference among different cultivars and also little difference between the male plants and the female plants.

28

Observations on leaf morphology of male and female Actinidia chinensis plants The study revealed that the ratio of guard cell length to width signicantly discriminated male and female plants. Thus the ratio of guard cell length to width could be used to effectively distinguish male plants from female plants both in seedlings and mature plants.

There was no stoma at the upper leaf surface in the 8 cultivars of A. chinensis. Each of the stomas in the lower leaf surface was enclosed by two kidney-shaped guard cells. Measurement of the guard cells showed that appearance of the guard cells was closely related to sexual distinction. The ratios of guard cell length to their width was greater than 3 in all the 4 male cultivars, and the ratio was less than 3 in all the 4 female cultivars (Table 2). Similar phenomenon was also present in the seedling plants germinated from the seeds of Heping No. 1 Bangzen No. 1 and Wuzhi No. 3 Fengxion No. 1 (Table 3). Thus, the ratio of guard cell length to width could be used as a reliable marker to distinguish male plants and seedlings from female plants and seedlings.
Table 2. Length and width of the guard cells of A. chinensis Cultivar Length (m) Width (m) Length / width Bangzen No. 1 31.6 9.1 3.5 Zhaoxia No. 3 28.9 6.2 4.7 Fengxion 27.8 9.1 3.1 Heping Hongyang 18.4 5.6 3.3 Heping No. 1 22.7 8.9 2.6 Miliang No. 1 19.1 7.8 2.5 Wuzhi No. 3 31.1 10.9 2.9 Heping Hongyang 20.1 6.9 2.9 Table 3. Length and width of the guard cells of A. chinensis seedlings Cross combination Length Width Length / width (m) (m) Heping No. 1 Bangzen No. 1 () 26.73.4 7.51.1 3.60.3 Heping No. 1 Bangzen No. 1 () 24.12.4 8.90.7 2.70.2 Wuzhi No. 3 Fengxion No. 1 () 26.11.7 7.80.2 3.40.2 Wuzhi No. 3 Fengxion No. 1 () 24.90.7 8.70.9 2.90.1

Acknowledgements
The work is supported by the Scientic Fund of Guangdong Province (No. 8151022501000010). No conict of interest is declared.

References
Chen, X.L., H. Liang and Z.W. Xie, 2008. Cluster analysis of fruit and leaf characters from 11 species of Actinidia. Journal Anhui Agricultural Sciences, 36(35): 15408-15410 (in Chinese). Chen, X.L., Z.W. Xie and H. Liang, 2005. Genetic diversity of Actinidia in Heping County of Guangdong Province. Advance in Actinidia Research, Beijing: China Science Press, 148-157 (in Chinese). Cui, Z.X. 1993. China Kiwifruit. Jinan: Shangdong Science and Technology Press, p. 6-66. (in Chinese) Gong, W.L. 1995. Direct identication of male and female plants of Populus talassica. Tianjin Agriculture and Forestry Science and Technology, 2: 40-41. (in Chinese) Huang, H.W. 2009. History of 100 Years of domestication and improvement of kiwifruit and gene discovery from genetic introgressed populations in the wild. Chinese Bulletin Botany, 44 (2): 127142. Huang, H.W., J. Gong and S.M. Wang, 2000. Genetic diversity of Actinidia. Biodiversity Science, 8(1): 49-54. (in Chinese) Huang, H.W., J. Li, P. Lang and S.M. Wang, 1999. Systematic relationships in Actinidia as revealed by cluster analysis of digitized morphological descriptors. Acta Hort., 498: 71-78. Li, J.Q., X.W. Li and D.D. Soejarto, 2007. Actinidiaceae. In: Flora of China. Vol.12. Z.Y. Wu, P.H. Raven, D.Y. Hong (Eds.), Beijing: Science Press & Saint Louis, Missouri: Missouri Botanical Gardens. pp. 334-360. Li, R.L., L.D. Lu, W.J. Gao, S.F. Li and Q. Wang, 2006. Advances in sex identication of dioecious plants. Guihaia, 26(4): 387-391. (in Chinese) Lian, Y.S., S.G. Lu and S.K. Xie, 2000. Biology and Chemistry of Hippophae. Langzhou: Gansu Science and Technology Press, p. 176-184. (in Chinese) Liu Y.L., Z. Li, P.F. Zhang, Z.W. Jiang and H.W. Wang, 2006. Spatial genetic structure in natural populations of two closely related Actinidia species (Actinidiaceae) as revealed by SSR analysis. Biodiversity Science, 14(5): 421-434. (in Chinese) Olah, R., E. Masarovicova and J. Samj, 1997. Anatomical and morphological parameters of leaves and leaf petioles of Actinidia deliciosa. Biologia Plantarum, 39(2): 271-280. Yang, M.X. 2010. Cytomorphological Observation on Sex Differentiation of Actinidia Chinensis. MS degree thesis, Zhongkai University of Agriculture and Engineering, (in Chinese). Yang M.X., H. Liang and S.D. He, 2009. Research progress in sex differentiation and identication of Actinidia. Journal Zhongkai University Agriculture Engineering, 22(1): 57-60. (in Chinese)
Received: February, 2011; Revised: August, 2011; Accepted: September, 2011

Discussion
It has long been thought that A. chinensis and A. deliciosa belong to different species (Li et al., 2007). However, many studies suggested that the cultivars of these two taxa overlapped in their morphological characters and molecular markers (Chen et al., 2008; Chen et al., 2005; Huang et al., 2000; Huang et al., 1999; Cui.1993). Now it is believed that these two taxa are different varieties of the same species, A. chinensis Planch var. chinensis and A. chinensis Planch var. deliciosa (Huang, 2009). Some differences were present in the cultivars between var. chinensis and var. deliciosa, and also present in the plants between male and female (Yang et al., 2009; Liu et al., 2006; Olah, 1997). Based on this study, the relative petiole length of male var. chinensis was longer than the female one, while that of male var. deliciosa was shorter than the female one. In both varieties, the ratio of petiole length to petiole diameter of the female plants was less than the matched male plant. The male petioles were relatively longer and thinner. Since leaf appearance of kiwifruit plants is quantitative character easy to be affected by nutritional, environmental and physiological factors, it is difcult to distinguish male plants from female ones according to the leaf appearance.

Journal

Journal of Applied Horticulture, 14(1): 29-32, 2012

Appl

Growth and foliar nutrient concentration response of Clerodendrum thomsoniae to increasing fertilization
Karen I. Davis, Carl E. Niedziela Jr.1*, Brian E. Whipker2 and Muchha R. Reddy Department of Natural Resources and Environmental Design, North Carolina Agricultural and Technical State University, Greensboro, NC 27411, 1Departments of Biology and Environmental Studies, Elon University, Elon, NC 27244, 2Department of Horticultural Science, Box 7609, North Carolina State University, Raleigh, NC 27695-7609. *E-mail: cniedziela@elon.edu

Abstract
The growth response, root substrate environment, and foliar nutrient concentrations of clerodendrum were evaluated in a range of fertilizer concentrations. A green-leaf selection of clerodendrum was grown for 129 days using a complete fertilizer containing micronutrients at concentrations of 50, 100, 200, 300 and 400 mg L-1 N. Shoot length and dry weight; root substrate electrical conductivity (EC); and foliar N, P, K, Cu, and Mn levels increased with increasing fertilizer concentration, while root substrate pH and foliar Mg and S decreased. The response of foliar Ca, Fe, Zn, and B concentrations to fertilizer concentration was not signicant. Although clerodendrum grown with 100 to 400 mg L-1 N had similar foliar N, P, and K concentrations by mean separation, foliage was lighter green at 100 mg L-1 N; thus 200 mg L-1 N is recommended because it provided adequate fertility without excessive shoot growth. Key words: Bleeding glory-bower, glory tree, fertilizer, nitrogen, phosphorous, potassium, Clerodendrum thomsoniae Balf.

Introduction
Clerodendrum thomsoniae is a vine grown commercially in hanging baskets (Dole and Wilkins, 2005). The trade and scientic literature contains a range of suggestions for fertilizing C. thomsoniae, however, recommendations based on replicated trials are unavailable. Sanderson et al. (1990) fertilized weekly with 20.0N-8.8P-16.6K (20N-20P2O5-20K2O) at 400 mg L-1 N. Alvensleben and Steffens (1989) used two applications of Ca(NO3)2 alternated with one application of 10.0N-5.2P-12.4K (10N-12P 2O 5-15K 2O) at 100 mg L -1 N. Wendzonka (1978) recommended applying an acidic fertilizer, such as 28.0N-7.9P6.6K (28N-18P2O5-8K2O) at 431 mg L-1 N weekly. Beck (1975) proposed applying a complete fertilizer at 200 mg L-1 N at each irrigation for the rst three weeks after potting then reducing the concentration to 100 mg L-1 N. Fertilization with 200 mg L-1 N beyond three weeks would delay owering. Koranski (1976) reported iron chlorosis when the root substrate pH exceeded 6.3. von Hentig (1987) reported injury of clerodendrum roots by high soluble salts and recommended maintaining root substrate between pH 5.5 and 6.5 to avoid Fe deciency. In a recent study, Davis et al. (2011) described additional disorder visual symptoms and established foliar nutrient concentrations in a variegated-leaf selection of clerodendrum grown to induce N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B deciencies and B toxicity. To increase growth efciency and plant quality, growers monitor and manage root substrate pH and electrical conductivity (EC) and foliar nutrient concentrations to provide adequate, but not excessive, levels of essential elements. Therefore, this study was conducted to evaluate the growth response, root substrate environment, and foliar nutrient concentrations of clerodendrum in a range of fertilizer concentrations.

Materials and methods

This fertilizer concentration experiment was conducted in the double layer polycarbonate Reid Greenhouse at North Carolina A&T State University in Greensboro, NC (36 north latitude) from 7 March to 13 July 2007. Rooted stem cuttings of a greenleaf selection of clerodendrum were transplanted with two plants per 19.05 cm diameter azalea (2.6 L) pot on 7 March 2007. The root substrate was 4 peat : 1 perlite (v/v) amended with dolomitic limestone at 5.9 kgm-3 and Aquagro 200-G (Aquatrols, Paulsboro, NJ) at 111 gm-3. Plants were fertilized at each irrigation with one of ve liquid fertilizer levels (50, 100, 200, 300, and 400 mg L-1 N) using Excel 13-2-13 (The Scotts Co., Marysville, OH), which contained the following percentage analysis of nutrients: 13N-0.86P-10.8K-6Ca-3Mg-0.006B-0.028Cu-0.05Fe-0.028Mn0.0075Mo-0.028Zn. Plants were irrigated as needed using a drip system utilizing sump-pumps (Model 1A, Little Giant Pump Co., Oklahoma City, OK), and plants were harvested at 37, 69, 97, and 129 d after planting. Greenhouse day/night set-points were 24/20 C. At each harvest date, plant height (measured from the substrate level to the uppermost part of each plant) and shoot dry weight were recorded. The most recently matured leaves were also sampled from the third harvest (97 d). Sampled leaves were rinsed in tap water for 30 s, then rinsed in deionized water for 30 s, and nally dried at 70 C for 48 h. On each harvest date, the root substrate in each pot was dried at 70C for 96 h. The substrate was then broken up and a representive sample of 100 cm3 was mixed with 200 mL of deionized water and allowed to rehydrate for 1 h. The pH and electrical conductivity (EC) were then recorded using a Hanna Meter HI 9811 (Hanna Instruments, Woonsocket, RI).

30

Growth and foliar nutrient concentration response of Clerodendrum thomsoniae at 69 and 129 d at the two highest fertilizer concentrations tested (300 and 400 mg L-1 N) were similar to each other with respect to least signicant differences. Root substrate: The responses of root substrate pH to fertilizer concentration were best-fit to linear models at both 69 and 129 d after planting (Table 1). Root substrate pH decreased with increasing fertilizer concentration. Root substrate pH was generally maintained between 5.5 and 6.5 as recommended by von Hentig (1987), except at the highest fertilizer concentration (400 mg L-1 N) where it dipped below pH 5.4. Iron deciency symptoms were not visible in any of the treatments. However, foliage on plants grown at the two lowest fertilizer concentrations (50 and 100 mg L-1 N) were an overall lighter green in color. The response of root substrate EC to fertilizer concentration was best-t to a linear model at both 69 and 129 d after planting (Table 1). Root substrate EC increased with increasing fertilizer concentration; however, soluble salts did not become excessive enough to inhibit growth. Foliar nutrient concentrations: The mean concentrations of all the measured nutrients in the most recently matured leaves at 97 d after planting were greater than the levels identied by Davis et al. (2011) as decient. However, fertilization did effect the foliar nutrient concentrations. The response of foliar N, P and K concentrations were best-t to linear models (Table 2). However, by mean separation, the foliar N, P and K concentrations at 50 mg L-1 N from 13N-0.86P-10.8K were lower than the foliar concentrations of these nutrients at the four higher fertilizer concentrations (100, 200, 300, and 400 mg L-1 N). Although deciency symptoms were not observed with 50 or 100 mg L-1 N , the plants at the two lowest concentrations were overall lighter green in color. This suggests that 100 mg L-1 N fertilizer rate provided inadequate nutrition. The response of foliar Ca concentration to fertilizer concentration was not signicant as determined by analysis of varience (Table 2). The mean Ca concentration for all fertilization treatments was 2.07%.

Tissue analysis: Oven dried tissue was ground in a stainless steel Wiley Laboratory Mill Model 4 (Thomas Scientic, Philadelphia) to pass a 1 mm screen (20-mesh). A 1.25 g sample was combusted at 490 C for 6 h. The resulting ash was dissolved in 10 mL 6 N HCl and diluted to 50 mL with deionized water. Phosphorus, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B concentrations were determined by inductively coupled plasma emission spectroscopy (ICPOES; Perkin-Elmer Optima 3300DV, Norwalk, CT). Nitrogen was determined with a Perkin Elmer 2400 CHN elemental analyzer (Norwalk, CT) on 10 mg samples. All tissue analyses were conducted at the Analytical Services Laboratory, School of Agriculture and Environmental Sciences, NC A&T State University. Experimental design and statistical analysis: The experiment was a randomized complete block design with ve fertilizer treatments four sample dates six replications. Data were subjected to analysis of variance using PROC ANOVA (SAS Inst., Cary, NC). Data values for dependent variables with signicant analysis of variances were regressed using the PROC GLM procedure to determine the best-t linear and quadratic models. Terms of the model were based on a comparison of F values at =0.05.

Results and discussion

Although data were collected on growth and substrate parameters at four destructive harvests, similar trends were observed at all four harvests. Therefore, only data from 69 and 129 d are presented to avoid redundancy. Growth: The responses to fertilizer concentration for stem length and shoot dry weight were best-fit to linear models at both 69 and 129 d after planting (Table 1). Stem length increased with increasing fertilizer concentration. The differences among treatments were greater at 129 d than 69 d as reected in the steeper slope on the linear equation. Shoot dry weight also increased with fertilizer concentration. Although these relationships were linear, the stem lengths and shoot dry weights

Table 1. Effect of fertilizer application of 13N-0.86P-10.8K at ve concentrations (50, 100, 200, 300, and 400 mg L-1 N) on the stem length, shoot dry weight, substrate pH, and substrate electrical conductivity of green-leaf clerodendrum at 69 and 129 days after planting Fertilizer concentration mg L-1 N 50 100 200 300 400 LSD (0.05)z Lineary Quadratic a b0x b1x2 r2 CV
z y

Stem length (cm) 69 days 129 days 59.3 64.0 71.2 88.0 96.2 19.1 <0.0001 0.8396 52.8 0.109 0.45 21 119.8 106.2 141.8 178.2 193.8 37.8 <0.0001 0.7429 95.2 0.251 0.49 23

Shoot dry weight (g) 69 days 129 days 8.9 10.7 14.7 17.6 16.4 4.9 0.0009 0.1292 8.64 0.0239 0.33 33 26.8 31.2 45.3 79.9 88.6 14.0 <0.0001 0.7622 13.8 0.193 0.83 21

pH 69 days 6.12 5.93 5.68 5.48 5.35 0.07 <0.0001 0.0040 6.17 -0.00217 0.92 2.5 129 days 6.35 6.10 5.68 5.53 5.38 0.12 <0.0001 <0.0001 6.38 -0.00272 0.87 2.4

Electrical conductivity mS cm-1 69 days 129 days 0.39 0.60 1.14 1.88 2.43 0.31 <0.0001 0.5811 0.0326 0.00598 0.90 21 0.42 0.77 1.46 1.75 2.24 0.28 <0.0001 0.1159 0.261 0.00509 0.87 19

LSD values are for comparing between fertilizer concentrations. The equation coefcients for either the linear (y = a + b0x) or quadratic (y = a + b0 x + b1x2) regression models are provided for whichever model had the lowest signicant Pr>F. The coefcient of determination (r2) and coefcient of variation (CV) were calculated for the best t model (n = 6). When the linear and quadratic models had the same level of signicance; the equation, r2, and CV are given for the simplest model (linear).

Growth and foliar nutrient concentration response of Clerodendrum thomsoniae

31

Table 2. Effect of fertilizer application of 13N-0.86P-10.8K at ve concentrations (50, 100, 200, 300, and 400 mgL-1 N) on foliar N, P, K, Ca, Mg, S, Cu, Fe, Mn, Zn, and B concentrations of green-leaf clerodendrum at 97 days after planting Fertilizer (mg L-1 N) 50 100 200 300 400 LSD (0.05)z Lineary Quadratic a b0x b1x2 r2 CV
z y

N 3.00 4.24 4.20 4.33 4.45 0.36 0.0001 0.0063 3.41 0.00302 0.42 12

P 0.53 1.09 1.17 1.34 1.41 0.32 0.0003 0.0961 0.670 0.00209 0.37 32

K (%) 2.47 3.30 3.22 3.30 3.58 0.49 0.0028 0.3154 2.70 0.00226 0.28 0.0028

Ca 2.14 2.29 2.15 1.82 1.94

NS

Mg

Cu 2.9 3.0 3.0 3.0 4.2 0.7 0.0029 0.0265 2.60 0.00295 0.28 20

Fe 88 93 104 108 111

NS

Mn (mg kg ) 46 57 68 109 123 28 <0.0001 0.6972 32.5 0.230 0.66 27

-1

Zn 12 18 15 18 18
NS

B 39 48 43 41 49
NS

0.86 0.30 0.92 0.32 0.78 0.24 0.64 0.24 0.60 0.22 0.12 0.04 <0.0001 <0.0001 0.9479 0.3834 0.953 3.22 -0.000922 -0.000283 0.51 0.47 16 15

LSD values are for comparing between fertilizer concentrations. The equation coefcients for either the linear (y = a + b0x) or quadratic (y = a + b0 x + b1x2) regression models are provided for whichever model had the lowest signicant Pr>F. The coefcient of determination (r2) and coefcient of variation (CV) were calculated for the best t model (n = 6).

The response of foliar Mg concentration was best-t to a linear model with foliar Mg concentration decreasing with higher fertilizer concentration (Table 2). The decrease in foliar Mg concentration with increasing fertilizer concentration 100 mg L-1 N was presumably due to an anatagonism by the monovalent cation macronutrients (Robson and Pitman, 1983). Foliar Mg concentrations at the three lower fertilizer concentrations (50, 100, and 200 mg L-1 N) were signicantly higher than the foliar Mg concentrations at 300 and 400 mg L-1 N. Although Mg deciency symptoms were not observed in this study, Mg deficiency symptoms have been observed during commercial clerodendrum production in NC (B. Whipker, personal observation). The response of foliar S concentration was best-t to a linear model with foliar S concentration decreasing with higher fertilizer concentration (Table 2). The decrease in foliar S concentration was presumably due to an anatagonism by the monovalent anion macronutrients. Foliar S concentrations at the two lowest fertilizer concentrations (50 and 100 mg L-1 N) were signicantly higher than the foliar S concentrations at 200, 300 and 400 mg L-1 N. The response of foliar Cu concentration was best-t to a linear model with foliar Cu concentration increasing with fertilizer concentration (Table 2). The foliar Cu concentrations at 50, 100, 200, and 300 mg L-1 N were signicantly lower than the foliar Cu concentration at 400 mg L-1 N. The response of foliar Mn concentration was best-t to a linear model with foliar Mn concentration increasing with fertilizer concentration (Table 2). The foliar Mn concentrations at 50, 100, and 200 mg L-1 N, respectively were lower than the foliar Mn concentrations at 300 and 400 mg L-1 N. The response of foliar Fe, Zn, and B concentrations to fertilizer concentration were not signicant as determined by analysis of variance (Table 2). The mean Fe, Zn, and B concentration for all treatments were 101, 16, and 44 mg kg-1, respectively. The results from this study contribute to understanding the fertilization of clerodendrum. Stem length and shoot dry weight of the greenleaf selection increased linearly with fertilizer concentration. At

97 d after planting, the lowest mean nutrient concentrations for the most recently matured leaves in the green-leaf selection for all nutrients were above the levels previously reported. Nutrients that increased with increasing fertilizer concentration (N, P, K, Cu and Mn) were above the critical minimum value in plants grown using 50 mg L-1 N. Although, clerodendrum grown at the four highest fertilizer rates had similar foliar N, P and K concentrations at 97 d, foliage on plants grown at 100 mg L-1 N were an overall lighter green in color. In plants grown at 400 mg L-1 N, nutrients that decreased with increasing fertilizer concentration (Mg and S) were above the level previously reported in plants showing foliar deciency symptoms, but lower than the other treatments at the two highest fertilizer rates (300 and 400 mg L-1 N). These results would indicate that among the concentrations tested, 200 mg L-1 N provided adequate mineral nutrition without excessive growth.

Acknowledgements
The authors gratefully acknowledge nancial assistance from the Opt-Ed Program at North Carolina Agricultural and Technical State University (NCA&TSU) and Agricultural Research Programs at NCA&TSU (Evans-Allen Funds) and North Carolina State University (Hatch Funds). Disclaimer : Mentioned trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by NCA&TSU or NCSU, and does not imply approval to the exclusion of other products or vendors.

References
Alvensleben, R.V. and M. Steffens, 1989. Clerodendrum thomsoniae. Grtnerbrse und Gartenwelt, 50: 2445-2447. Beck, G.E. 1975. Preliminary suggestions for the culture and production of clerodendrum. Ohio Florists Assn. Bull. 547. Ohio State Univ., Columbus, OH. Davis, K.I., C.E. Niedziela Jr., M.R. Reddy, B.E. Whipker and J.M. Frantz, 2011. Nutrient disorder symptomology and foliar concentrations of Clerodendrum thomsoniae. J. Plant Nutr., 34(7): 1079-1086. Dole, J.M. and H.F. Wilkins, 2005. Floriculture Principles and Species. Second Edition. Prentice-Hall, Upper Saddle River, NJ.

32

Growth and foliar nutrient concentration response of Clerodendrum thomsoniae

von Hentig, W.U. 1987. Clerodendrum thomsoniae, In: KulturKartei Zierpanzenbau. Verlag, Parey, Berlin and Hamburg. p. C-3 Wendzonka, P. 1978. Clerodendrum hanging baskets. Focus Floricult., 6(2): 6-7.
Received: May, 2011; Revised: August, 2011; Accepted: December, 2011

Koranski, D.S. 1976. Growth and owering of Clerodendrum thomsonae Balif. Ph.D. Diss., University of Wisconsin, Madison. 1976. 155 pp. Robson, A.D. and M.G. Pitman, 1983. Interactions between nutrients in higher plants. In: Encyclopedia of Plant Physiology, Volume 15A: Inorganic Plant Nutrition. A. Lauchli and R.L. Bieleski (Eds.). Springer-Verlag, NY. pp.147-180. Sanderson, K.A., W.C. Martin Jr. and J. McGuire, 1990. New application methods for growth retardants to media for production of clerodendrum. HortScience, 25(1): 125.

Journal

Journal of Applied Horticulture, 14(1):33-39, 2012

Appl

Inuences of severe water stress on photosynthesis, water use efciency and proline content of almond cultivars
Kazem Barzegar, Abbas Yadollahi*, Ali Imani1 and Noorollah Ahmadi Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 1 Department of Horticultural Science, Seed and Plant Improvement Institute (SPII), Karaj, Iran. *E-mail: yadollah@modares.ac.ir

Abstract
Using drought tolerant almond cultivars under arid and semiarid regions such as Iran is important factor affecting production yield, especially in rainfed orchards. To evaluate responses of almond cultivars to drought stress under eld condition, the experiment was carried out on six commercial cultivars namely Azar, Marcona, Mission, Nonpareil, Sahand, and Supernova. Net photosynthesis rate (Pn) and water use efciency (WUE) data during three stress periods indicated that Pn decreased in stress treatments, but WUE increased under stress treatments. The highest Pn occured in Azar in July and August, and the highest WUE was recorded in Sahand and Supernova. Leaf abscission in Sahand was very high and Supernova had no signicant abscission. Leaf relative water content (RWC) showed a downward trend from June to August. In Azar, Nonpareil and Supernovacultivars, RWC resulted from severe stress treatment had close relationship with RWC in well-watered treatment. This result may be due to osmoregulation in leaves of stressed plants. So these cultivars could keep high water content in their leaves and tolerate severe drought stress conditions than other investigated cultivars. The highest and lowest proline accumulation was observed in the leaves of Marcona and Sahand, respectively; both Marcona and Sahand were sensitive to drought stress than Supernova which showed medium proline accumulation. In almond, accumulation of proline in response to longer interval between irrigation is a general trait and cannot be used as indicator for dening the tolerant trees. In general, Supernova and Azar showed best response under drought stress. Key words: Drought tolerance, Pn, WUE, RWC, Proline, Prunus dulcis Mill.

Introduction
Water plays essential role in many physiological processes during plant life. Water absorbed by the roots is translocated to aerial portions of the plant and lost to the atmosphere via pores of the stomatal apparatus. Transpiration ratio is used to assess the plant effectiveness in regulating water loss which is essential for CO2 uptake consumed in photosynthesis. An unbalance in water ow can result in water decits and faulty functioning of numerous cellular processes (Taiz and Zeiger, 2006). Photosynthesis and subsequently cell growth and productivity are severely affected by water potential and its components. Clearly, drought is one of the most common environmental factors affecting plant growth and productivity in arid and semiarid zones. WUE increases under reduced-water supply and this response to drought has an adaptive signicance (Raviv and Blom, 2001). Lower gas exchange reduces carbon assimilation in leaves under drought stress condition. Limitation of photoassimilates reduces vegetative growth and severely retard the development of plant reproductive organs (Boyer, 1970; Gehrmann, 1985; Singer et al., 2003). Genotypic differences in drought tolerance have been observed for various crops (Bota et al., 2001). Drought tolerant species have a capacity to maintain relatively high rate of photosynthesis under drought stress (Gu et al., 1999). Romero et al. (2004) observed that during pre-harvest period, photosynthesis rate of drought-stressed almonds was lower than

that of unstressed plants under control conditions, whereas during subsequent recovery their photosynthesis rate was the same as or even better than that of control plants. Leaves of drought tolerant plants such as almond, olive, and some of forest trees can reach extremely low values of leaf RWC: 75-80%, before losing turgidity (Hinckley et al., 1980; Lo Gullo and Salleo, 1988; Larsen et al., 1989). One of the most widely distributed compatible osmolytes in higher plants is the amino acid proline. Accumulation of proline in plant leaves is related to irrigation intervals, as proline content in leaves increased sharply under drought stress and remained at higher levels during the stress condition (Al-Karaki et al., 1996). The aim of this study was to characterize response of six almond cultivars to drought stress based on their photosynthesis activity, water use efciency and proline accumulation in leaves.

Material and methods

Plant material and eld condition: The experiments were carried out from May to September 2010 on six almond (Prunus dulcis Mill) cultivars viz., Azar, Marcona, Mission, Nonpariel, Sahand, and Supernovoa. These cultivars were raised in the open led almond collection at Karaj-Iran (35 55 N, 50 54 E, 1312.5 m a.s.l.), under semi dry climate on calcareous soils (Table 1 and 2). Experimental design: Almond cultivars Azar, Marcona, Mission, Nonpariel, Sahand, and Supernovoa grafted on

34

Inuences of severe water stress on almond cultivars RWC (%) = [(FW DW) / (TW DW)] 100.
45-120 Sandy 2-16 4-18 62-98 7.5-7.8 0.8 9.8 0.1 2.3 54.5 10 5.3 24.7

Table 1. Soil properties at experimental site Soil characteristics* Depth (cm) 0-20 20-45 Sandy clay Soil texture Sandy clay loam loam Clay (%) Silt (%) Sand (%) pH EC CaCO3 (%) Organic C (%) Available P (mg kg-1) Available K (mg kg-1) Humidity percent in W.P Humidity percent in F.C Saturation humidity (%) 20-36 26-36 30-60 7.4-7.8 2.4 10.4 0.6 11 225 18.5 9.5 35.3 22-30 22-38 32-60 7.6-7.9 0.7 14.5 0.27 2.6 86 17 9 35.5

Proline: Proline was measured according to the method described by Bates et al. (1973). In order to measure proline content changes within 30-day period of severe stress conditions, samples of leaves were collected from new shoots before irrigation on June 27th, July 27th and August 27th. Leaf sample of 0.5 g was used for extraction. A Shimadzu spectrophotometer (UV- 160A) was used for reading absorption at 520 nm. Statistical analysis: The statistical analysis was performed using Microsoft Excel (Microsoft ofce version 2007 package) and SAS software (SAS Institute Inc, 1990) and means were compared using Duncans Multiple Range Test at P 0.01 (DMRT).

Results
Photosynthesis rate (Pn): As depicted in Fig. 1, at rst stage, Nonpareil with 3.93 mol CO2 m-2s-1 and Mission with 3.93 mol CO2 m-2s-1 showed highest and lowest net photosynthetic rate, respectively. But in July and August, Azar (5.2 and 2.9 mol CO2 m-2s-1) exhibited high Pn than other investigated cultivars. Pn of Sahand and Nonpareil (1.4 and 0.67 mol CO2 m-2s-1) were lowest at July and August, respectively.
2.0 62

*Soil and Water Research Institute, Karaj, 2010 Table 2. Climatic conditions at experimental site, Karaj, 2010 Month Apr May Jun Jul Aug Sept Oct Average temperature (C) 17.2 20.3 26.6 32.4 28.4 25.6 19.6 Rainfall (mm) 35.4 28.8 Relative humidity percent 58 68 0.2 58 0 57 1.2 66 0 63

bitter almond seedling were planted at 5 5 m space in 2007. This experiment was arranged in a complete randomized design with three replications. Trees were subject to two irrigation regimes. In optimal irrigation regime (control), trees were irrigated to maintain 90100% of soil water capacity (SWC); while in reduced irrigation regime (stress treatment), soil moisture was maintained at a level of about 10% of SWC. Both irrigation regimes were applied to the plants throughout their growing season. Rainfall was stopped at May 10th at the experimental site. The period of drought stress started since May 31th. Trees under stress and normal conditions were irrigated monthly and 7 days intervals, respectively. Measurements: Net photosynthetic rate (Pn), photosynthetically active radiation (PAR) and transpiration rate (E) were measured by a LCA4 (ADC BioScientic Ltd., England) in the eld at 10-12 AM at three stages during the stress period (25 June, 25 July, and 25 August). Water use efciency was evaluated using following formula (Molden 1997): WUE = Pn (mol CO2 m-2s-1) /E (mmol H2O m-2s-1). Relative Water Content (RWC) : Midday relative water content (RWC) was measured on June 25, July 25 and August 25 (beginning, middle and the end of drought stress) at the same time of day. To determine leaf fresh weight (FW), 5 leaves of each treatment/genotype were detached and weighed by electronic balance, Then leaves were hydrated until saturation (constant weight gained) for 48 h at 4 oC in darkness (turgidity weight: TW). Leaves were then dried in an oven at 105 oC for 24 h for determining dry weight (DW). RWC was calculated according to Filella et al. (1998):

Fig. 2 display Pn in studied cultivars in the well-watered treatments. The highest Pn was recorded on 25th June for all cultivars. At this stage Pn of Marcona (12.54 mol CO2 m-2s-1) was the highest and Nonpareil (7.12 mol CO2 m-2s-1) was the lowest. In July and August, Azar with 8.83 and 6.45 mol CO2 m-2s-1 showed highest rate of photosynthesis, while the lowest rate was recorded in Mission cultivar. In general, Pn decreased from June to August in each cultivars.. Water use efciency (WUE): There was signicant difference between cultivars in measured WUE. As shown in Fig. 3, Sahand at rst and the third stage showed the highest WUE (3.74 and 4.14 mol CO2 mmol H2O1, respectively), while WUE of Supernova was high (5.54 mol CO2 mmol H2O1) at second stage (25th July). During that period, Mission had the lowest WUE (1.05, 1 and 1.56 mol CO2 mmol H2O1). In the three stages and also between cultivars WUE was different. But in the well-watered treatments, WUE at the rst recording stage was several time more than that of later stages as well as stressed conditions (Fig. 4). Highest WUE was recorded in Azar at the 1st stage, in Supernova at the 2nd and 3rd stage whereas lowest WUE was in Nonpareil, Azar and Mission at 1st, 2nd and 3rd stage, respectively. Relative water content (RWC): The data showed that the leaf relative water content ranged between 65 to 85% in all cultivars. RWC in the well-watered treatments was higher than RWC resulted in stress treatments. Although in the second stage of the experiment, except for Marcona, there werent signicant differences in RWC between well-watered and stressed plants. But at 1st and 3rd stage, measured RWC of Sahand, Marcona and Mission had signicant differences between treatments. Overall Marcona showed signicant differences and Azar, Nonpareil and Supernova had no differences between treatments (Fig. 5). Proline: Irrigation intervals had a signicant effect on proline content of the leaves. Although proline content in the leaves at the beginning of the experiment was similar, it increased sharply in

Inuences of severe water stress on almond cultivars

35

Fig.1. Pn of severe stressed treatments on 25th June, 25th July and 25th August

Fig. 2. Pn of normal irrigated treatments on 25th June, 25th July and 25th August

Fig. 3. WUE at three stages (25th June, 25th July and 25th August, named 1, 2 and 3) in the severe stressed plants.

36

Inuences of severe water stress on almond cultivars

Fig. 4. WUE at three stages (25th June, 25th July and 25th August named 1, 2 and 3) in the well-watered plants

Fig. 5. RWC of 6 different almond cultivars during 3 months water stress. Star (*) shows the signicant differences between well irrigated treatment compared with severe water stress treatments. Each point is the average of three replications and vertical bars indicate SEM.

Inuences of severe water stress on almond cultivars

37

Fig. 6. Leaf proline content of 6 different almond cultivars during 3 months water stress. Star (*) shows the signicant differences between well irrigated treatment compared with severe water stress treatments. Each point is the average of three replications and vertical bars indicate SEM.

the longer (30 days) irrigation treatment and remained at higher levels during the experiment. There were signicant differences between severe stress and well-watered treatments during 3 stages (Fig. 6). The proline content of samples showed an increase in stress treatment during the rst 30 day of the experiment. At the second stage of sampling proline content showed very high value in severe stress than well-watered treatments (Fig. 6).

Discussion
Drought can seriously reduce Pn and productivity of crops. Plant physiological responses such as photosynthesis and transpiration depend on the rapidity, severity and duration of drought (Lawlor and Cornic, 2002; Ramachandra Reddy et al., 2004). The decline in photosynthesis during the early stages of water stress

38

Inuences of severe water stress on almond cultivars made in response to soil water deficit and low leaf water potentials were also observed (Torrecillas et al., 1988; Klein et al., 2001). The study of the patterns of leaf water relations and gas exchange activity is a good physiological approach for analyzing the optimum water use by plants (Hsiao, 1993) and can provide fundamental information on plant responses to irrigation treatments. This imbalance is more intense under high evaporative demand and/or severe soil water decit (Yadollahi et al., 2011). Changes in water content under severe drought stress applied initially (from 1st June to 29th June, RWC once measured at 25 June) indicate signicant difference between well-watered and severe stress treatments. So due to lack of sufcient available water in the soil, osmotic adjustment in leaves was low in stressed treatments. At the second stage of RWC monitoring (25th July) two experimental treatments showed similar results. There were no signicant differences in RWC between stressed and wellwatered treatments in the most of cultivars. This result can be due to osmotic adjusting in leaves of stressed plants. The leaf RWC showed a downward trend from June to August. RWC in the leaves of severe stress treatment had close relationship with well-watered treatment in Azar, Nonpareil and Supernova. So these cultivars were able to keep high water content in their leaves and tolerate severe drought stress conditions than other experimental cultivars. Proline has been primarily known as a protective and osmoregulatory agent for plant cells under environmental stresses (Shevyakova et al.,1985). Its production rate in plants with different stress tolerance is not similar and even proline content could be lower in more tolerant plants (Shevyakova et al., 1985). Although proline concentration of almond leaves increased with longer irrigation intervals, differences between varieties were not signicant to characterize and screen almond plants for drought tolerance based on their proline production. In our experiment severe stress also caused increased proline amount in leaves of all of cultivars; although, it was different between cultivars (Fig. 6). The highest proline accumulation was observed in the leaves of stressed Marcona but it was least in Sahand. Both Marcona and Sahand were sensitive to drought stress than Supernova. It would be concluded that accumulation of proline in response to long time interval between irrigation is a general trend and cannot be used for dening the more tolerant almond trees. Our data showed that cultivars Azar and Supernova are resistant cultivars under severe drought stress. Based on their high WUE and Pn, these cultivars use osmotic adjustment to keep high amount water in their leaves during drought stress conditions. Proline cannot be used as an indicator in determining drought resistant cultivars. Applying 3 time irrigation in semiarid regions such as Karaj, Iran situations will be enough, without any signicant effect on performance of mentioned cultivars.

Fig. 7. Percentage of leaf abscission of cultivars under severe drought stress condition at the end of experiment.

was attributed to a combination of stomatal and non-stomatal factors, whereas a further decline in photosynthetic activity under more severe water stress conditions could be due to nonstomatal factors such as biochemical changes induced under severe stress. According to Williams et al. (1999), the decrease in photosynthetic rate at low water availability was primarily caused by stomata closure. Our results showed that decreased photosynthesis efciency could be related to interference in stomatal conductance. After 20 days of drought stress, Nonpareil and Sahand showed a higher rate of photosynthesis than other cultivars. At second stage, Pn of Azar, Marcona and Mission increased, but Pn of Sahand, Nonpareil and Supernova decreased. At 25th August, Pn decreased slightly in all stressed treatments. This change indicate adaptation to severe drought stress. WUE increased under reduced-water supply. Since WUE increases as the soil water supply decreases, this response to drought has an adaptive signicance (Raviv and Blom, 2001). Comparison of WUE data of well-watered and stressed treatments at three stages of growing season has shown that WUE increased after drought stress. Because of low dehydration and high photosynthesis during early season, WUE in control plants was very high. But simultaneous increase in temperature and transpiration inuenced water loss. This condition leads to drastic reduction in WUE during season in well-watered plants. But in the plants under severe stress conditions, WUE was higher than control plants except at rst stage. Sahand and Supernova had low and high WUE, respectively. Considerable leaf abscission in Sahand and negligible abscission in Azar and Supernova was observed (Fig .7). Overall Supernova and Azar were superior than the other stressed cultivars. Klein et al. (2001) suggested an overriding control on stomatal aperture exerted by low relative humidity, high vapor pressure decit and temperature in almond leaves, although adjustments

Acknowledgment
The authors acknowledge the nancial support provided to this project by Tarbiat Modares University (TMU) through Grant Number TMU 88-12-132.

Inuences of severe water stress on almond cultivars

39

References
Al-Karaki, R.N., R.B. Clark and C.Y. Sullivan, 1996. Phosphorous nutrition and water stress effects on proline accumulation in sorghum and bean. J. Plant Pyhsiol., 148: 745-751. Bates, L.S., R.P. Waldren and I.D. Teare, 1973. Rapid determination of free proline for water stress studies. Plant Soil, 39: 205-207. Bota, J., J. Flexas and H. Medrano, 2001. Genetic variability of photosynthesis and water use in Balearic grapevine cultivars. Ann. Appl. Biol., 138: 353-361. Boyer, J.S. 1970. Leaf enlargement and metabolic rates in corn, soybean, and sunower at various leaf water potentials. Plant Physiol., 46: 233-235. Filella, I., J. Llusia, J.O. Pin and J.U. Pen, 1998. Leaf gas exchange and uorescence of Phillyrea latifolia, Pistacia lentiscus and Quercus ilex saplings in severe drought and high temperature conditions. Environ. Exp. Bot., 39: 213-220. Gehrmann, H. 1985. Growth, yield and fruit quality of strawberries as affected by water supply. Acta Hort., 171: 463-469. Gu, Z., J.J. Hu, J.L. Wen and S.Q. Wang, 1999. A study on adaptability of maple to drought stress. J. Northwest Forest., Col., 14: 1-6. Hinckley, T.M., F. Dubme, A.R. Hinckley and H. Richter, 1980. Water relations of drought hardy shrubs: osmotic potential and stomatal reactivity. Plant Cell Environ., 3: 13 l-140. Hsiao, T.C. 1993. Growth and productivity of crops in relation to water status. Acta Hort., 335: 137-147. Klein, I., G. Esparza, S.A. Weinbaum and T.M. DeJong, 2001. Effects of irrigation deprivation during the harvest period on leaf persistence and function in mature almond trees. Tree Physiol., 21: 10631072. Larsen, F.E., S.S. Higgins and A. Al wir, 1989. Diurnal water relations of apple, apricot, grape, olive and peach in an arid environment. Sci. Hort., 39: 211-222. Lawlor, D.W. and G. Cornic, 2002. Photosynthetic carbon assimilation and associated metabolism in relation to water decits in higher plants. Plant Cell Environ., 25: 275-294. Lawlor, D.W. 2002. Carbon and nitrogen assimilation in relation to yield: mechanisms are the key to understanding production systems. J. Exp. Bot., 53: 773-787.

Lo Gullo, M.A. and S. Salleo, 1988. Different strategies of drought resistance in three Mediterranean sclerophyllous trees growing in the same environmental conditions. New Phytol., 108: 267-276. Molden, D. 1997. Accounting for Water Use and Productivity. SWIM Paper 1. International Irrigation Management Institute. Colombo, Sri Lanka. Ramachandra Reddy, A., K. Viswanatha Chaitanya and M. Vivekandan, 2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J. Plant Physiol., 161: 1189-1202. Raviv, M. and T.J. Blom, 2001. The effect of water availability and quality on photosynthesis and productivity of soilless-grown cut roses. Sci. Hort., 88: 257-276. Romero, P., J.M. Navarro, F. Garca and P.B. Ordaz, 2004. Effects of regulated decit irrigation during the pre-harvest period on gas exchange, leaf development and crop yield of mature almond trees. Tree Physiol., 24: 303-312. SAS Institute. 1988. SAS/STAT Users Guide: Release 6.03 ed. SAS Inst., Inc., Cary, NC. Shevyakova, N.I., B.P. Strogonov and I.G. Kiryan, 1985. Metabolism of polyamines in NaCl-resistant cell lines from Nicotiana sylvestris. Plant Growth Regul., 3: 365-369. Singer, S.M., Y.I. Helmy, A.N. Karas and A.F. Abou-Hadid, 2003. Influences of different water stress treatments on growth, development and production of snap bean (Phaseolus vulgaris L.). Acta Hort., 614: 605-611. Torrecillas, A., M.C. Ruiz-Sanchez, F. del Amor and A. Leon, 1988. Seasonal variations on water relations of Amygdalus communis L. under drip irrigated and non irrigated conditions. Plant Soil, 106: 215-220. Taiz, L. and E. Zeiger, 2006. Plant Physiology. Fourth Edition. Sinauer Associates. Sunderland, MA. 764 p. Williams, M.H., E. Rosenqvist and M. Buchhave, 1999. Response of potted miniature roses (Rosahybrida) to reduced water availability during production. J. Hort. Sci. Biotechnol., 74: 301-308. Yadollahi, A., K. Arzani, A. Ebadi, M. Wirthensohn and S. Karimi, 2011. The response of different almond cultivars to moderate and severe water stress in order to screen for drought tolerance, Sci. Hort., 129: 403-413.
Received: December, 2011; Revised: January, 2012; Accepted: February, 2012

Journal

Journal of Applied Horticulture, 14(1): 40-46, 2012

Appl

Selection of resistant source to early blight disease in tomato among the Solanum species
A.K. Singh, N. Rai*, R.K. Singh, Major Singh, R.P. Singh1, Smita Singh and Satyandra Singh Indian Institute of Vegetable Research, P.B.- 5002, P.O.-BHU, Varanasi, (U.P.) India - 221005, 1Udai Pratap Autonomous P.G. College, Varanasi, (U.P.) India- 221005. *E-mail: nrai1963@gmail.com

Abstract
Resistance to early blight (EB) disease of tomato caused by Alternaria solani was assessed by examining various parameters of the disease progress. For this study twenty three diverse tomato genotypes were screened under replicated trials for over three years (2007-2009) using articial inoculation under controlled conditions as well as under natural epidemics at Indian Institute of Vegetable Research, Varanasi, UP, India. Tested genotypes showed signicant difference in their response to A. solani and disease severity. Area under disease progress curve (AUPDC) was positively correlated with percent disease index (PDI) and negatively with resistance. Of the 23 genotypes, only two i.e. EC-520061 (Solanum habrochaites) and H-88-78-1 (S. lycopersicum) were highly resistant (PDI < 5.0; AUDPC < 200 and r value 0.12) for EB disease under eld and glasshouse environments. Characterization using molecular markers also indicated their resistance. It was concluded that there are signicant differences between resistant and susceptible tomato lines against EB disease and some of the lines should be considered resistant rather than tolerant. Hence, the choice of resistant lines can be utilized in future breeding programmes for development of early blight resistant/tolerant cultivars of tomato. Key words: Early blight, Alternaria solani, Solanum habrochaites, resistant, AUDPC

Introduction
Tomato (Solanum lycopersicum L. syn. Lycopersicon esculentum Mill.) is one of the most important vegetables grown worldwide. Besides several pests and diseases, the early blight (EB) caused by Alternaria solani (Ellis & Martin) Sorauer, is one of the most damaging foliar diseases capable to cause serious yield losses worldwide (Sherf and MacNab, 1986; Pandey et al., 2003; Peralta et al., 2005) and is highly correlated with the disease intensity (Dater and Mayee, 1985). The control measures include crop rotation with non-hosts for several years, routine applications of chemical fungicides, and the use of disease-free germplasm (Madden et al., 1978; Sherf and MacNab, 1986). Fungicide treatment is generally the most effective control measure but may not be effective under certain weather conditions favorable for epidemics (Herriot et al., 1986). Conditions that favour early blight include heavy rainfall, high humidity, and relatively high temperatures (Chaerani and Voorrips, 2006). Due to health and environment concern, growers are looking for safer alternative approaches. Resistant cultivars are potentially the most economical and effective way to combat the disease as they can keep away and/or extend the fungicide spray intervals (Madden et al., 1978; Shtienberg et al., 1995; Keinath et al., 1996). Cultivars highly resistant to early blight are not known in cultivated tomato (Chaerani et al., 2007); however, some disease tolerant cultivars/lines are available (Nash and Gardner, 1988; Gardner and Shoemaker, 1999; Upadhyay et al., 2009). Moreover, much vegetable cultivar evaluation focus on yield and fruit quality attributes with less emphasis on disease tolerance. The molecular markers identied and characterized for early blight resistance in wild tomato species using interval mapping and selective genotyping approaches is immense benecial for

breeding programmes (Foolad et al., 2000). One approach is the identication of candidate resistance genes using polymerase chain reaction (PCR) technology and sequence information from known disease resistance genes (R genes). Hence, it is imperative to screen tomato cultivars/lines against early blight disease caused by A. solani to identify sources of resistance having desirable qualitative and quantitative characteristics which could be utilized in traditional breeding approaches. Keeping this in view the objectives of the study were to screen tomato lines possessing good horticultural traits under natural epidemics and articially inoculated controlled conditions, and to assess the disease progress with time and its relationship with the age of tomato plant.

Materials and methods

Twenty three phenotypically diverse tomato genotypes including 20 S. lycopersicum and three wild species (S. pimpinellifolium S. glandulossum and S. habrochaites) were obtained from the gene bank of Indian Institute of Vegetable Research, Varanasi, India (Table 1). All the 23 genotypes were screened under eld and articial (screen house) condition for three consecutive years (2007-09) at the research farm of IIVR, Varanasi. For eld evaluation, twenty one days old seedlings were transplanted in sick-plots (2.5 2.0 m). Twenty plants per plot of each tomato line were maintained with three replications in a randomized complete block design (RCBD) at row to row and plant to plant spacing of 60 and 45 cm, respectively. Experiments were repeated twice during the study in eld. No fungicide or other chemical was used throughout the study period. All standard practices were adopted to raise a good tomato crop. In eld tests, EB severity was assessed in terms of percent defoliation and the average portion of necrotic leaf area on the plant (Horsfall and Barrat, 1945).

Selection of resistant source to early blight disease in tomato among the Solanum species

41

Table 1. Description of tomato genotypes tested against early blight disease caused by Alternaria solani Genotypes Source Species Growth habit Selection based on the character Agata-30 NBPGR, New Delhi, India S. lycopersicum Determinate Fruit size and number of fruits Agata-32 NBPGR, New Delhi, India S. lycopersicum Determinate Fruit size and number of fruits CO-3 TNAU, Coimbator, India Determinate Light green large fruit & good yield S. lycopersicum DT-10 IARI, New Delhi, India Determinate Fruit oval, normal standing plants, more number of fruits S. lycopersicum DVRT-2 IIVR, Varanasi, UP., India Determinate Large fruit size and round shape S. lycopersicum EC-520061 NBPGR, New Delhi, India S. habrochaites Indeterminate Wild, long, aromatic, high TSS and more number of fruits. EC-521080 NBPGR, New Delhi, India S. pimpinellifolium Indeterminate Wild, long, high TSS and more number of fruits FEB -2 NBPGR, New Delhi, India S. lycopersicum Determinate Round fruit FLA-7171 John Scott, Univ. Florida Determinate Medium fruit size and round in fruit shape S. lycopersicum FLA-7421 John Scott, Univ. Florida Determinate Large round fruit, attractive S. lycopersicum H-24 HAU, Hissar, India Determinate Normal in fruit size and good yield S. lycopersicum H-86 IIVR, Varansi, UP, India Determinate Large fruit and good yield S. lycopersicum H-88-78-1 IIVR, Varansi, UP, India Indeterminate Cultivated , high TSS and good yield S. lycopersicum H-88-78-2 IIVR, Varansi, UP, India Indeterminate Cultivated, round and hard fruits. S. lycopersicum H-88-87 IIVR, Varansi, UP, India Indeterminate cultivated line, good yeild S. lycopersicum KT-15 IARI, New Delhi, India Determinate More number of fruits and medium size fruits S. lycopersicum PKM-1 TNAU, Coimbator, India Determinate Bunchy type plants and attractive dark green colour S. lycopersicum Punjab Chhuhara PAU, Ludhiana, India Determinate Long fruit, good yield and attaractive S. lycopersicum Roma IARI, New Delhi, India Determinate Medium fruit size and oval in shape, with light green colour S. lycopersicum SEL-7 HAU, Hissar, India Determinate Large fruit size and round in shape S. lycopersicum Shalimar-2 SKAT, Srinagar, J&K, India S. lycopersicum Determinate Medium and oval fruit Sikkim Local WIR-3928 Sikkim, India NBPGR, New Delhi, India S. lycopersicum S. glandulossum Indeterminate Cultivated, long plant height and good yeild Indeterminate Wild, high TSS Sum of all ratings x 100 Total number of observations maximum rating grade

For screen house studies, a total of 230 earthen pots were lled with sandy loam soil (soil, sand and farm yard manure in 2:1:1 ratio) and kept in randomised block design, maintaining 10 plants for each tomato line. The screen house was maintained at 2220C and more than 95% relative humidity (RH). Experimental designs and replications were same for other screen house experiments which were also repeated twice during the study. The pure culture of A. solani was raised on potato dextrose broth (PDB) in 500 mL conical asks. Three hundred mL of PDB was autoclaved in a vertical type autoclave at 15 psi for 20 minutes. After sterilization the asks were observed for a period of 48 h to ensure no contamination. Inoculation with pure culture of tested fungus was carried out under aseptic condition followed by incubation for two weeks at 261C. Both fungal mat and broth were thoroughly mixed with the help of electric mixer and grinder. An inoculum concentration of 125 cfu/mL was maintained and uniformly sprayed on four week old tomato plants (Pandey et al., 2003) maintained in 6 inches diameter plastic pots containing approximately 1 kg sandy loam soil. The plants were kept in screen house and regularly fertilized with Hoagland solution. All the observations were recorded at 24 h interval based on the disease severity on zero to ve (0-5) point scales: 0-Free from infection; 1-One or two necrotic spots on a few lower leaves of plant; 2-Few isolated spots on leaves, covering nearly 5%10% of the surface area of the plant; 3-Many spots coalesced on the leaves, covering 25% of the surface area of the plant; 4-Irregular, blighted leaves and sunken lesions with prominent concentric rings on the stem, petiole, and fruit, covering 4050% of the surface area; 5-Whole plant blighted, leaves and fruits starting to fall. The disease severity in terms of PDI, AUDPC and r were recorded as per Pandey et al. (2003).

PDI=

The mean value of PDI was calculated and host plant reaction was categorised on the following basis: 0-5%-highly resistant (HR); 5.1-12%-resistant (R); 12.1-25%-moderately resistant (MR); 25.1-50%-moderately susceptible (MS); 50.1-75%-susceptible (S) and >75%-highly susceptible (HS). The area under disease progress curve (AUDPC) was calculated using the following formula:
AUDPC = [(
i =1 n 1

X i +1 + X i ) *(t2 t1 ) 2

Where, Xi is the disease index expressed as a proportion at the ith observation; ti is the time (days after planting) at the observations and n is the total number of observations and rate of infection (r) was calculated as: 1 n (1 n1 ) r= log e 2 t 2 t1 n1 (1 n 2 ) Where, t1 is the time (days) during the rst observation; t2 is the time during the second observation; t2 - t1 is the time interval between two observations and subsequently; n1 is the PDI at time t1; and n2 is the PDI at time t2. Data analysis: Data on disease severity and development were pooled and subjected to ANOVA using SPSS Ver. 12.0. Differences between treatments were determined by Duncans Multiple Range Test at 5% signicance level. PCR reaction for screening through molecular markers: Total genomic DNA was extracted using modied CTAB protocol (Doyle and Doyle, 1990) from young leaves of six tomato genotypes that showed highly resistant, resistant and highly susceptible disease reactions. The master mix consisted of 1.0 L dNTPs (containing 10 mM each dNTPs), 2.0 L MgCl2 (25.0 mM

42

Selection of resistant source to early blight disease in tomato among the Solanum species had the highest PDI and AUDPC followed by Punjab Chhuhara. The PDI values were highest for highly susceptible lines under both natural (70%) and articially inoculated controlled (79%) environments. Susceptible lines were easily infected and had extremely high AUDPC (1661 to 3240) under naturally infected field conditions. Negative correlation between the AUDPC and degree of resistance against pathogen was noted whereas a positive correlation was recorded between AUDPC and PDI which seems to increase with susceptibility. Most of the susceptible lines were determinate except H-88-78-2 and H-88-87 which were moderately susceptible, however, few of them (Punjab Chuhara, DVRT-2 and Roma) had qualitative and quantitative desirable characters and are already under cultivation. The disease progress was also studied for both susceptible and resistant lines for two different time intervals (7 to 28 days and 45 to 90 days, respectively). For this study three lines from each group: EC-520060, H-88-78-1 and WIR 3928 of resistant group and CO-3, Punjab Chhuhara and Sel-7 of susceptible group were examined. Both groups of inoculated plants had distinctly different curves (Fig. 2a and b) at both time intervals. Initially, the curve seemed to increase rapidly followed by a gradual increase in case of susceptible lines. However, in case of resistant lines it was very slow and did not have any peak. It shows that high degree of resistance does not allow the causal organism to infect the plant rapidly. It was observed that the disease progress under both environment were almost similar at the initial stage but differed at the later stage as disease progressed in due course of time. The total area under disease increased gradually from resistant to highly resistant and susceptible to highly susceptible lines. Line Sel-7 reaction to A. solani was different from other lines during initial days of observation (Fig. 2a). The disease severity of susceptible lines increased with time, up to 90 days (Fig. 2b). In another set of experiments, the effect of tomato plants age on PDI and AUPDC was also studied (Fig. 3a and b). It is notable that young plants (45 days) were less susceptible to A. solani infection compared to older (60 days) plants. The mean PDI and AUDPC values for most tested genotypes were much higher in older plants with highest value in case of CO-3 (up to 68.77 and 1247.25, respectively) compared to young plants (34.86 and 470.03, respectively). The apparent infection rate between two subsequent observations was more informative than the total infection rate in terms of disease spread. It was low in resistant varieties compared to susceptible ones. A positive correlation was observed between the disease incidence and age of plant under both conditions. The SSR primers were used for polymorphic survey between resistant and susceptible lines with monomorphic or multiple bands products. These SSR markers were discarded and polymorphic primers repeated twice. Under present study, 15 SSR primers (SSR 104, SSR 108, SSR 112, SSR 124, SSR 156, SSR 210, SSR 218, SSR 226, SSR 241, SSR 304, SSR 308, SSR 310, SSR 316, SSR 350 and SSR 356) gave polymorphism against EC-520061 and were monomorphic against other tomato lines viz., Punjab Chhuhara, H-88-78-1, Sel-7, CO-3 and WIR-3928 revealed that EC-520061 is most polymorphic source to use for development of resistant lines/varieties against early blight in tomato (Fig. 4).

MgCl2 ), 0.75 L Taq polymerase, 2.5 L 10X reaction buffer, 1.0 L of forward and reverse primer and 1.0 L of genomic DNA. The PCR programme followed one cycle of pre denaturation at 94 0C for 4 min, 35 cycles of denaturation at 94 0C for 1 min, annealing at 50-55 0C for 1 min, extension at 72 0C for 1 min, and one nal extension cycle at 720C for 10 min. The amplication products were separated on 2.0% agarose gels (Bredemeijer et al., 2002). The gels were analyzed using a gel documentation system- Alpha Imager (Alpha Innotech, USA). A large set of SSR primers were surveyed to nd the polymorphic primers between the appropriate resistant and susceptible tomato lines.

Results
Tomato lines were screened for early blight disease under natural epidemic and screen houses by artificial inoculation under controlled conditions and the disease severity was compared in both the environments. Of 23 screened tomato lines (Table 1), four lines with indeterminate growth habit [EC-520061 (S. habrochaites), EC-521080 (S. pimpinellifolium), H-88-78-1 (S. lycopersicum) and WIR-3928 (S. glandulossum)] were recorded resistant against early blight under both the environments on the basis of PDI, AUDPC and r value. The lines EC-520061 and H-88-78-1 were highly resistant under both environments while response of EC-521080 and WIR-3928 differed with the environments (Fig. 1-3). Among these four lines, three (EC520061, EC-521080 and WIR-3928) belong to wild species whereas H-88-78-1 is a cultivated line but derivative of S. habrochaites f. glaboratum, which seems to be the rst report that shows high resistance against early blight disease. The mean value of PDI, r and AUPDC of all tested materials were higher under controlled conditions compared to open eld condition (Fig.1a, b and c). The mean PDI of highly resistant lines (EC520061 and H-88-78-1) was <5.0% (i.e. 0.0 and 4.75%). For resistant lines (EC-521080 and WIR-3928) the value of PDI was between 5.1 to 12% (Fig. 1a). It is noticeable that all the four resistant lines had indeterminate growth, and remained disease free for a longer period in the eld without fruit or with few fruits. Being cultivated, H-88-78-1 line has some desirable characters that can be utilised for further improvement in the breeding programs. Likewise the value of infection rate was also recorded minimum in case of highly resistant lines followed by resistant lines (0.00 to 0.11). The infection rate was positive under both (eld and screen house) conditions as the disease severity clearly increased between the rst and last observation. The mean infection rate of all tested materials was higher under controlled conditions compared to eld condition (Fig.1b). Under natural infection in eld the AUDPC of the highly resistant variety EC520061 was 0.0 and it was 23.1 when inoculated with A. solani under articially controlled conditions. In case of other resistant lines it ranged from 137.5 to 206.4 under natural epidemics and 158.9 to 303.8 under articially inoculated controlled conditions. Tomato lines that showed susceptible and highly susceptible reaction had extremely high AUDPCs, under both environments (Fig.1c). Most of the tested lines with good yield characteristics were either susceptible or highly susceptible under both the conditions. Susceptible lines were easily recognised by symptoms like infected stem, leaves, calyx, petiole etc. and the infection gradually increased leading to progressive increase in PDI with each observation. On the basis of disease parameters, line CO-3

Selection of resistant source to early blight disease in tomato among the Solanum species A

43

Fig. 1. Effect of early blight disease of tomato caused by A. solani on different genotypes under naturally infested eld and articially inoculated conditions (A) percentage disease index, (B) apparent infection rate and (C) area under disease progress curve. Vertical lines represent standard error.

Area under disease progression curve

Discussion
It is reported that the indeterminate growth habit of tomato crops has negative relation to biotic and abiotic stresses. It may be due to their continuous growth and physiological or biochemical

changes in plants life cycle. In our study, the highly resistant (EC520061 and H-88-78-1) and resistant (EC-521080 and WIR-3928) lines also had indeterminate growth habit. These results conrm the disease relation with plant growth habit in most accessions as reported by Pandey et al. (2003). Lines, H-88-87, H-88-78-2

44

Selection of resistant source to early blight disease in tomato among the Solanum species

A B

Fig. 2. Disease progress curve for early blight disease of tomato caused by A. solani on selected susceptible and resistant lines (A) Under articially inoculated conditions up to 28 days and (B) Under naturally infected eld condition up to 90 days.

Fig. 3. Relationships between age of tomato genotypes with (A) Percentage disease index of early blight disease of tomato caused by A. solani (B) Area under disease progress curve of early blight disease of tomato caused by A. solani. Vertical lines represent standard error.

and Sikkim Local have indeterminate growth habit but showed moderately susceptible disease reaction, due to their slow growth habit compared to other indeterminate accessions at each stage. Whereas, Agata-30, FEB-2, FLA-7171, FLA-7421, H-24, PKM-1 and Roma have determinate growth habit and able to hold-up during an epidemic period without being much affected by the disease and also possess acceptable qualitative and quantitative characters (Foolad et al., 2000).

Manual screening under eld condition is a primary procedure for screening but controlled (articial) condition screening is considered as better alternative for getting reliable information for EB disease (Pandey et al., 2003). Various methods have been used for evaluation of plants for disease resistance by many workers (Kalloo and Banerjee, 1993; Pandey et al., 2003; Upadhyay et al., 2009). Natural epidemics of early blight are strongly inuenced by environmental conditions, even though severe disease appears every year in northern India (Pandey et al.,

Selection of resistant source to early blight disease in tomato among the Solanum species

45

Fig. 4. Lane 1 DNA Ladder 100bp (Fermentas), Lane 2-48, genotypes in sequence (WIR 3928, Punjab Chhuhara, H-88-78-1, EC-520061, CO-3 and Sel-7). Primers (SSR 226, SSR 210, SSR 308, SSR 310, SSR 316, SSR 124, SSR 112 and SSR 356)

2003). The conditions (average temperature 22 C and humidity around 90%) which favour the disease are prevalent in the study area. A major hindrance in breeding tomatoes for early blight resistant has been the screening process due to its limitations such as environmental conditions. Field screening can often be carried out only once a year, such limitations would restrict breeding process (Foolad et al., 2000). The effectiveness of each method may vary by species to species and by application. Therefore, it is reasonable here to use more than one method. In the present study, PDI, AUDPC and r were used to evaluate and compare disease severity of tested tomato lines. Periodic observations of PDI are essential to assess the pathogenic reaction on a particular line. Based on a single observation, it is difcult to evaluate the disease severity and pathogen reaction of the same line at later stages (Pandey et al., 2003). The present ndings also conrm that the PDI and AUDPC are important for controlled environment and apparent infection rate are important for natural screening programmes. The disease severity initially progress slowly but accelerate as plant matures. The highly resistant and resistant lines EC-520061 (S. habrochaites), EC-521080 (S. pimpinellifolium), WIR-3928 (S. glandulossum) and H-88-78-1 (S. esculentum) have indeterminate growth and according to Pandey et al. (2003), these resistance lines are stable and an indication of resistance against early blight disease caused by A. solani. An area under disease progress curve which refers to as the signature of an epidemic, represent the integration of all the host, pathogen and environmental factors during epidemics (Campbell and Madden, 1990). Screening under articial condition is more informative than natural conditions and for inoculation only mycelia cultures of the pathogen were used. The result conrms that the degree of susceptibly to A. solani infection increases with the age of plants and the inuence of climatic factor is minimal because of the controlled condition. In inoculated plants, disease incidence always increased during the initial disease development (7-28 days after inoculation) and thereafter subsequently up to 90 days. Disease developed quickly during natural epidemics at the time of fruit set and infected leaves quickly defoliated. It further increased if either irrigation or rain prevailed in the eld. Similar results were recorded on tomato (Pandey et al., 2003; Upadhyay et al., 2009) and other crops (Rotem, 1994; Pandey and Pandey, 2002). Our study has established that the parameters e.g., PDI, r and AUDPC are important and useful to screen tomato lines against early blight caused by A. solani. Many PCR based tomato markers have been developed and

mapped, and are publicly available from a variety of sources (e.g. http://www.sgn.cornell.edu/, http://hornbill.cspp.latrobe. edu.au/ssrdiscovery.html). These resources enabled screening a set of genome-wide markers to identify polymorphic markers distinguishing genotypes previously screened in field and glasshouse test (articial screening). If the markers are informative, they may be also applied on the population developed between the crosses. (resistant vs. susceptible). The selected primers are useful for QTL mapping against early blight reaction. Majority of these anchor markers such as SSR 108 (500bp) which exhibited polymorphism in the survey had previously associated with early blight mapping (Chaerani, 2000) indicating their usefulness for mapping in other populations also. It is notable in the present ndings that cultivated tomato line H88-78-1 (S. esculentum) performed equally well as wild relatives, EC-520061 (S. harbochaites) in suppressing the disease. In view of the above, it is concluded that the genotypes EC-520061 and H-88-78-1 may be used as donor parents to develop early blight disease resistance/tolerant cultivars/hybrids and could be of immense importance for further traditional or molecular breeding programs.

Acknowledgements
The authors gratefully acknowledge Director, IIVR, Varanasi, U.P., India for providing tomato genotypes and necessary facilities during this study. Thanks are also due to technical and supporting staff of the centre for their assistance in conducting the experiments.

References
Bredemeijer G.M.M., P.J. Cooke, M.W. Ganal, R. Peeters, P. Isaac, Y. Noordijk, S. Rendell, J. Jackson, M.S. Rder, K. Wendehake, M. Dijcks, M. Amelaine, R. Wickaert, L. Bertrand and B. Vosman, 2002. Construction and testing of a microsatellite database containing more than 500 tomato varieties. Theor. Appl. Genet., 105: 1019-1026. Campbell, C.L. and L.V. Madden, 1990. Introduction to Plant Disease Epidemiology. Wiley, New York. Chaerani, R. and R.E. Voorrips, 2006. Tomato early blight (Alternaria solani ): The pathogen, genetics, and breeding for resistance (Review). J. Gen. Plant Path., 72: 335-347. Chaerani, R., R. Groenwold, P.S. Roeland and E.Voorrips, 2007. Assessment of early blight (Alternaria solani ) resistance in tomato using a droplet inoculation method. J. Gen. Plant Path., 73: 96103. Datar, V.V. and C.D. Mayee, 1985. Breeding for early blight resistance in tomato. Ind. Phytopath., 33: 151.

46

Selection of resistant source to early blight disease in tomato among the Solanum species
Pandey, K.K. and P.K. Pandey, 2002. Incidence of cowpea foliar blight caused by Pseudocercospora cruenta in relation to weather factors. Ind. Phytopathol., 55: 206-209. Pandey, K.K., P.K. Pandey, G. Kalloo and M.K. Banerjee, 2003. Resistance to early blight of tomato with respect to various parameters of disease epidemics. J. Gene. Plant Pathol., 69: 364371. Peralta, I.E., S. Knapp and D.M. Spooner, 2005. New species of wild tomatoes (Solanum section Lycopersicon: Solanaceae) from Northern Peru. Syst. Bot., 30: 424-434. Rotem, J. 1994. The genus Alternaria: Biology, Epidemiology and Pathogenicity. APS Press, St. Paul, MN, pp. 264-272. Sherf, A.F. and A.A. MacNab, 1986. Vegetable Diseases and Their Control. Willey, New York. pp. 728. Shtienberg, D., D. Blachinsky, Y. Kremer, G. Ben-Hador and A. Dinoor, 1995. Integration of genotype and age-related resistance to reduce fungicide use in management of Alternaria diseases of cotton and potato. Phytopathol., 85: 995-1002. Upadhyay, P., P.C. Singh, B. Binha, M. Singh, R. Kumar and K.K. Pandey, 2009. Source of resistance against EB (Alternaria solani). Ind. J. Agric. Sci., 79: 9.
Received: September, 2011; Revised: December, 2011; Accepted: January, 2012

Doyle, J.J. and J.L. Doyle, 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15. Foolad, M.R., N. Ntahimpera, B.J. Christ and G.Y. Lin, 2000. Comparison of eld, greenhouse, and detached-leaet evaluations of tomato germplasm for early blight resistance. Plant Dis., 84: 967-972. Gardner, R.G. and R.B. Shoemaker, 1999. Mountain supreme early blight resistant hybrid tomato and its parents, NCEBR-3 and NCEBR-4. HortScience, 34: 745-746. Harriot, A.B., F.L. Jr. Haynes and P.B. Shoemaker, 1986. The heritability of resistance of early blight in diploid potatoes. Amer. J. Potato., 63: 229-232. Horsfall, J.G. and R.W. Barrat, 1945. An improved grading system for measuring plant diseases. Phytopathol., 35: 655. (Abstr.). Kalloo, G. and M.K. Banerjee, 1993. Early blight resistance in Lycopersicon esculentum Mill. transferred from L. pimpinellifolium (L.) Mill. and L. hirsutum f. glabratum Mull. Gartenbauwissenschaft. 58: 238-240. Keinath, A., V.B. DuBose and P.J. Rathwell, 1996. Efcacy and economics of three fungicide application schedules for early blight control and yield of fresh-market tomato. Plant Dis., 80: 1277-1282. Madden, L., S.P. Pennypacker and A.A. MacNab, 1978. FAST, a forecast system for Alternaria solani on tomato. Phytopathol., 68: 1354-1358. Nash, A.F. and R.G. Gardner, 1988. Heritability of tomato early blight resistance from Lycopersicon hirsutum, PI 126445. J. Am. Soc. Hort. Sci., 113: 264-268.

Journal

Journal of Applied Horticulture, 14(1): 47-50, 2012

Appl

Water requirement of pomegranate (Punica granatum L.) plants upto ve year age
D.T. Meshram*, S.D. Gorantiwar, H.K. Mittal, N.V. Singh and A.S. Lohkare National Research Center on Pomegranate, Shelgi Bypass, NH-9, Solapur-413 006. Maharashtra, India. *E-mail: gomesh1970@rediffmail.com

Abstract
The study was carried out to estimate reference crop evapotranspiration, develop crop coefcient, area factors and estimates of pomegranate evapotranspiration for Pune region of Maharashtra. The crop coefcient values were estimated on weekly basis from the concept of shaded area approach that is widely used for the deciduous crops. Shaded area was estimated at 12.00-13.00 h with the help of specially prepared plywood board of different sizes with grid marking of size 20 x 20 cm for 5 randomly selected pomegranate trees each from 2 orchards of different ages. The values of water to be applied to pomegranate plantation spaced at 4.5 x 3 m and irrigated by the drip irrigation system of 90 % efciency were estimated for 1st, 2nd, 3rd, 4th and 5th year of pomegranate orchard for Ambe Bahar, Mrig Bahar and Hasta Bahar. The values of water to be applied presented in this paper would be useful for the appropriate irrigation water management of pomegranate. Key words: Pomegranate, reference crop evapotranspiration (ETr), actual evapotranspiration (ETc), crop coefcient (kc), area factor (Fa)

Introduction
Pomegranate (Punica granatum L.) is one of favourite fruit crops of tropical and sub-tropical regions. It is a high value crop and has great economic signicance (Levin, 2006; Jalikop, 2007; Holland et al., 2009). India is the largest producer of pomegranate (Jadhav and Sharma, 2007). It is commercially cultivated in Maharashtra followed by Karanataka, Andhra Pradesh and Gujarat. The total pomegranate production in the world is around 15 lakhs tonnes out of which India produces 8.28 lakhs tonnes, but exports only 33.4 thousand tones (Holland et al., 2009). At present, around more than 1.27 lakh ha area is covered in India under pomegranate cultivation of which around 1 lakh ha is in Maharashtra alone, producing about 67 % of total Indian production. The productivity level is still low (<6.7 t/ha) in India as compared to the major pomegranate producing countries like Israel, Iran, Spain, China, (>40 t/ha) etc. (Holland and BarYaakov, 2008). In Maharashtra, pomegranate is commercially cultivated in the regions of Solapur, Nasik, Ahmednagar, Pune, Sangli, Satara, Dhule, Aurangabad, Latur and Osmanabad. In the pomegranate growing area of Maharashtra, water is scarce and hence, there is a need to apply water according to water requirement of the crop. The water requirement of crop depends on age, season, location and management strategies (Allen et al.,1998). There are many methods reported in the literature to estimate reference crop evapotranspiration. The important methods include: Penman, Modified Penman, Penman-Monteith, Hargreaves-Samani, Pan Evaporation, Blanney-Criddle, Radiation, Jensen-Haise, Priestly-Taylor, Thronthwaite and Christiansen. The excellent reviews on these methods have been provided by Doorenbos and Pruitt (1977) and Patil and Gorantiwar (2009). Some methods require huge data set but are considered accurate whereas other require less data and give

approximate value. FAO Penman-Monteith method (Allen et al., 1998) has been recommended as a standard. Therefore, in this study, estimates of ETc for pomegranate were made by PenmanMonteith method. Computation of water requirement needs the measurement of evapotranspiration (ETc). However, evapotranspiration is not easy to measure. Specic devices and accurate measurements of various physical parameters or the soil water balance in lysimeters are required to measure evapotranspiration (Doorenbos and Pruitt,1977). These methods are often expensive and demanding in terms of accuracy of measurements and can only be fully exploited by well trained research personal. Although, the methods are inappropriate for routine measurements, they remain important for the evaluation of ETr estimates obtained by indirect methods. However due to simplicity, indirect method use weather parameters for estimation of ETr. The ETc is then estimated by multiplying ETr with kc. The development of simple method to estimate seasonal kc for different crops, including woody, perennial, horticultural crops would be of great benet to the horticultural industry (Williams and Ayars, 2005). Hence, accurate estimation of ETr and kc are of paramount importance for proper irrigation scheduling. The paper presents the methodology used for determination of ETr, kc, WR and the weekly values of water to be applied in Ambe Bahar, Mrig Bahar and Hasta Bahar for the pomegranate orchards of Pune region of Maharashtra in India.

Materials and methods

Determination of kc values: The kc values vary with the crop growth stages and the age of the crop. The determination of kc values needs the measurement of ETc and the estimation of ETr. ETc values need to be measured with help of lysimeters or soil moisture studies. As the pomegranate is a widely spaced fruit crop

48

Water requirement of pomegranate (Punica granatum L.) plants upto ve year age effective root zone only. Hence water requirement in case of drip irrigation method was estimated by following equation: WR = ETc x Fa, Where, WR = water requirement (mm d-1), Fa = Area factor (fraction) Area factor: Area factor is the proportion of the effective root zone with respect to the total area. The area factor hence varies with the crop growth period and the age of the crop. In general, it has been reported that for most of the deciduous crops, the effective root zone area below the soil surface is the area occupied by the canopy above the soil. The canopy area is the shaded area at solar noon hour and was measured weekly for all the tress under experimentation. Area factor was computed by using following relationship: Fa = SA/AT, Where, Fa=Area factor, SA=Shaded area (m2), AT=Area occupied per tree (m2). Fa was calculated on weekly basis for the pomegranate trees up to the age of 5 years. Water to be applied: Water to be applied was estimated on weekly basis up to the age of 5th year by using the equation given bellow: WA = WR * A/eff, Where, WA = water to be applied to each tree (L d-1), A = area occupied by each tree (m2), eff. = efciency of the drip irrigation system (fraction)

and stabilizes after 4-5 years, it is required to grow pomegranate for 4-5 years in large lysimeters. Such type of experimental set up is very expensive and takes lot of time to generate the information, though accurate. Hence in this study it was proposed to develop the kc values with the help of shaded area approach that is adopted for many deciduous crops. For this purpose, two commercial pomegranate orchards (Mrig Bahar) of 1st to 5th years were selected from the Sangola Tehsil and from each orchard 5 of representative plants were randomly selected. The shaded area was measured at solar noon hour with the help of specially prepared plywood boards of 1.5 x 1.5, 2.5 x 2.5, 3.5 x 3.5 m sizes with grid marking of size 20 x 20 cm. The total numbers grids occupied by shaded area were measured on a weekly basis for each selected plant. The crop coefcient was then calculated by following equation developed for deciduous fruit crops (Williams and Ayars, 2005) kc=0.014x-0.08, Where, kc = Crop coefcient, x = Percentage of shaded area, (%) By using the above equation, the week wise crop coefcient values were developed for different phenological stages from June (Mrig Bahar) from 1st to 5th year age. The values so developed for Mrig Bahar were then appropriately transformed for Ambe and Hasta Bahar. Estimation of ETr: The Penman-Monteith method (Allen et al., 1998) was used for the estimating reference crop evapotranspiration by following equation: ETr=
900 0.408(Rn-G)+( T+273 ) u2 (es-ea) + (1+0.34 u2

Results and discussion

The average ETr values estimated by Penman-Monteith method are depicted in Fig. 1. It is revealed from the gure that, ETr is the highest in May (19-20 MW) and lowest in the month of December (49-52 MW). The weekly values of ETr are useful for obtaining the crop evapotranspiration of any crop for which the crop coefcient values are known. It is noted at this juncture that in lack of locally developed values of stage wise kc, the values proposed by FAO (Doorenbos and Pruitt, 1977) are being widely used. The mean yearly values of ETr as obtained by PenmanMonteith method are 1428.00 mm estimated for Pune region. The monthly values of SA and kc are presented in Table 1 for Mrig Bahar and transformed values for Ambe Bahar and Hasta Bahar for the pomegranate tree of different ages. It is apparent from the table that the shaded area increased from new leaf initiation to maturity period in the range from 0.84 to 10.60 m2. During harvesting period shaded area decreased from 10.60 to 7.25 m2 due to leaf drop, less amount of irrigation, removing of water sprout, luxur and harvesting of fruit. The monthly crop coefcient values of pomegranate tree of
50 45 40 35 30 25 20 15 10 5 0 1 4

Where, ETr = reference evapotranspiration (mmd-1), G = soil heat ux density (MJm-2d-1), Rn = net radiation (MJm-2d-1), T = mean daily air temperature (0C), = psychometric constant (kPa/0C), = slope of saturation vapour pressure function (kPa/0C), es = saturation vapour pressure at air temperature T (kPa), ea = actual vapour pressure at dew point temperature (kPa), u2 = average daily wind speed at 2 m height (m sec-1) This method needs the daily values of meteorological parameters viz., maximum temperature, minimum temperature, maximum relative humidity, minimum relative humidity, wind speed and sunshine hours. The daily records of these parameters were obtained from the Indian Meteorological Department, Pune region. The daily values of ETr were estimated for all years. Daily ETr values were summed up to obtain to weekly ETr values. Estimation of ETc: The weekly values of ETr and kc were used to obtain weekly values of ETc by following equation for Ambe Bahar, Mrig Bahar and Hasta Bahar for all the years. ETc = ETr x kc, Where, ETc= pomegranate evapotranspiration (mm d-1), ETr = reference crop evapotranspiration (mm d-1), kc = crop coefcient of pomegranate Water requirement: The water requirement by the surface irrigation methods is equal to the crop evapotranspiration estimated by the equation. However water requirement by the drip irrigation method is less than the water requirement of the surface irrigation methods as in drip irrigation method unlike in surface irrigation method, it is possible to apply water to the

7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 Tim e (w e e k) Fig. 1. Average weekly ETr values for Pune region from 1987 to 2009.

Average weekly ETr values (mm)

Water requirement of pomegranate (Punica granatum L.) plants upto ve year age different ages for Mrig Bahar shown in Table 1 can be used for the estimation of pomegranate evapotranspiration provided the values of reference crop evapotranspiration are known. According to important phenological stages of mature pomegranate tree (Table 2), the period of new leaf initiation to 10 % ground cover of tree is 21 days, the crop development period i.e. up to 60 to 80 % ground cover of the tree is 77 days, the maturity period is 56 days and harvesting period is 105 days. During the period from the new leaf initiation to crop development, the crop coefcient values increased from 0.22 to 1.10 and during maturity period, the kc values were around 1.2. At harvesting, the value of kc went on decreasing from 1.14 to 0.65 due to leaf drop, removing of water sprout, foliage breakdown and harvesting of fruits. The average shaded area per plant and wetted area for pomegranate tree are presented in Table 3. The water to be applied to the pomegranate plantation irrigated by surface irrigation methods can be calculated by using the ETr values (Fig.1) and kc values (Table 1). The water to be applied to the pomegranate plantation irrigated by drip irrigation method can be calculated by using the ETr values (see materials and methods) and kc and SA values (Table 1), if the efciency of the drip irrigation method and the area covered by the pomegranate tree are known. Usually the pomegranate is spaced at 4.5 x 3 m and the drip irrigation systems are designed for 90 % efciency. Hence, the
Table 1. Monthly shaded area (SA) and crop coefcient (kc) values of 1st to 5th year pomegranate trees for Mrig Bahar Month 1st SA July 0.84 August 1.00 September 1.16 October 1.28 November 1.37 December 1.49 January 1.66 February 1.60 March 1.47 April 1.56 May 1.65 June 1.79 kC 0.16 0.17 0.18 0.20 0.21 0.22 0.23 0.25 0.25 0.23 0.24 0.25 Age of pomegranate tree (year) 2nd 3rd 4th SA kC SA kC SA kC 1.68 0.22 1.35 0.13 1.72 0.14 2.43 0.25 4.09 0.21 4.82 0.26 3.30 0.32 7.53 0.48 8.32 0.54 4.05 0.41 9.54 0.83 10.30 0.91 4.13 0.49 9.63 1.06 10.44 1.13 3.44 0.51 8.21 1.08 9.41 1.15 2.65 0.44 7.14 0.94 8.12 1.05 2.01 0.35 6.11 0.78 6.85 0.92 2.01 0.29 5.54 0.68 6.41 0.79 2.16 0.29 5.72 0.65 6.57 0.74 2.34 0.30 5.94 0.67 6.73 0.77 2.50 0.32 6.14 0.69 6.88 0.78 5th SA kC 2.07 0.15 5.26 0.30 8.46 0.59 10.55 0.92 10.60 1.16 9.73 1.18 8.91 1.09 7.73 1.00 7.25 0.88 7.58 0.83 7.77 0.87 8.02 0.89

49

values of water to be applied to pomegranate in different seasons for different stations were estimated for the tree spacing of 4.5 x 3.0 m and drip irrigation efciency of 90 %. The values of water to be applied in liter/day on weekly basis are presented in Table 4 for Ambe, Mrig and Hasta bahars. However, this period can be up to 2 months depending on the climate and soils. After the stress period is over, it is necessary to bring the moisture content in the root zone to the eld capacity. For this purpose, it is proposed to operate the drip irrigation sysem continuously for 24 to 48 hours. The values in the table would be useful for irrigation scheduling of pomegranate by drip irrigation method. The values of reference crop evapotranspiration were estimated by the Penman-Monteith method and is presented in this paper on weekly basis for the Pune region and would be useful to estimate the pomegranate evapotranspiration, if the values of crop coefcient are known. The area factor values developed in this study would be useful to estimate the water requirement of pomegranate trees of different ages in combination with the crop coefcient values. The values of water to be applied to pomegranate (spaced at 4.5 x 3 m) irrigated by drip irrigation method (efciency=90%) on weekly basis for Pune region (Maharashtra) would be useful for the irrigation planning, optimum and efcient utilization of irrigation water.

Acknowledgements
The authors thank Shri. Prabakar Sadhasiv Chandane, a progressive farmer for providing pomegranate orchards and necessary help for experimentation. This work was supported by Ph.D. work at CTAE, MPUAT, Udaipur.

References
Allen, R.G., L.S. Pereira, D. Raes and M. Smith, 1998. Crop Evapotranspiration, Guideline for Computing Crop Water Requirements. FAO Irrigation and Drainage Paper 56. FAO Rome, Italy, pp. 300. Doorenbos, J. and W.O. Pruitt, 1977. Guidelines for Predicting Crop Water Requirement. FAO Irr. and Dran. Paper No. 24, FAO, Rome, Italy, pp.156. Holland, D. and I. Bar-Yaakov, 2008. The pomegranate: New interest in an ancient fruit. Chronica Hort., 48: 12-15. Holland, D., K. Hatib and I. Bar-Yaakov, 2009. Pomegranate: Botany, horticulture, breeding. Hort.Rev., 35: 127-191. Jadhav, V.T. and J. Sharma, 2007. Pomegranate cultivation is very promising. Indian Hort., 52: 30-31. Jalikop, S.H. 2007. Linked dominant alleles of inter-locus interaction results in a major shift in pomegranate fruit acidity of Ganesh and Kabul Yellow. Euphytica, 158: 201-207. Levin, G. M. 2006. Pomegranate (1st Edn), Third Millennium Publishing, East Libra Drive Tempe, AZ, pp. 13-120. Patil, P.D. and S.D. Gorantiwar, 2009. Probability analysis of weekly crop evapotranspiration of Rahuri region. Intl. J. Agr. Eng., 2: 68-71. William, L.E. and J.E. Ayars, 2005. Grapevine water use and the crop coefcient are linear functions of the shaded area measured beneath the canopy. Agr. For. Meteorol., 132: 201-211.
Received: April, 2011; Revised: November, 2011; Accepted: December, 2011

Table 2. Important phenological stages of matured pomegranate tree under Pune conditions Phenological stage Meteorlogical week Periods Initial Crop development Mid season Late season Rest period 31st to 33rd 34th to 44th 45th to 52nd 01st to15th 16th to 30th 21 days 77 days 57 days 105 days 105 days

Table 3. Year wise area factor of pomegranate tree Year Age of pomegranate tree Shaded area (m ) Area per plant (m2) Wetted area (Fraction)
2

1st 2.65 13.5 0.20

2nd 4.05 13.5 0.30

3th 5.40 13.5 0.40

4th 6.75 13.5 0.50

5th 8.10 13.5 0.60

50

Water requirement of pomegranate (Punica granatum L.) plants upto ve year age

Table 4. Water to be applied (L d-1 t-1) for 1st to 5th year pomegranate tree for Ambe Bahar, Mrig Bahar and Hasta Bahar M W 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1st 1.25 1.34 1.46 1.59 1.67 1.89 2.11 2.27 2.42 2.62 2.87 3.13 3.31 3.54 3.68 3.81 4.09 4.28 4.35 4.36 4.23 3.88 3.77 2.91 2.78 2.46 2.55 2.60 2.38 2.38 2.39 2.22 2.43 2.05 2.14 2.39 2.40 2.43 2.30 2.28 2.32 2.40 2.42 2.36 2.31 2.19 2.13 2.06 2.08 2.09 2.16 2.42 Ambe Bahar 2nd 3rd 4th 2.59 1.98 3.02 2.93 2.70 4.49 3.35 3.66 6.18 3.76 5.03 8.46 4.10 6.27 10.26 4.86 8.46 13.37 5.61 11.14 17.24 6.25 13.66 20.80 6.91 16.23 24.91 7.69 19.57 29.33 8.78 23.11 34.88 9.70 27.00 40.67 10.91 30.30 45.66 12.16 34.49 50.89 13.07 37.60 55.20 13.44 38.43 56.67 14.34 40.78 60.09 14.91 42.28 62.12 15.00 42.43 62.31 15.08 42.80 62.42 14.51 41.09 59.93 13.22 37.31 54.42 11.68 34.03 50.82 8.57 24.73 37.67 7.76 22.29 34.22 6.54 18.61 28.96 6.31 18.13 28.69 6.00 17.50 27.83 5.18 15.25 24.41 4.87 14.25 23.20 4.62 13.74 22.22 3.97 12.05 19.61 3.93 12.64 20.15 3.74 11.87 19.04 3.91 11.99 18.82 4.39 13.42 21.12 4.41 13.48 21.16 4.50 13.61 21.33 4.33 12.92 20.24 4.30 12.79 20.04 4.35 12.91 20.19 4.49 13.26 20.75 4.56 13.41 21.09 4.47 13.05 20.63 4.41 12.74 20.01 4.21 12.06 18.73 4.11 11.76 18.15 3.99 11.31 17.41 3.98 11.27 17.28 4.00 11.21 17.21 4.15 11.56 17.63 4.63 12.89 19.58 5th 3.39 5.48 7.65 10.43 12.63 16.01 20.55 24.37 28.50 33.58 38.66 44.56 49.90 56.97 61.83 63.39 67.28 69.61 69.88 70.07 67.33 61.19 56.52 42.20 38.59 32.83 33.08 32.43 29.01 27.95 27.29 24.46 25.22 22.18 23.07 25.82 25.82 25.96 24.61 24.33 25.08 25.70 25.90 25.19 24.56 23.28 22.53 21.61 21.57 21.56 22.12 24.68 M W 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1st 1.50 1.39 1.52 1.53 1.62 1.85 1.88 1.94 1.87 1.87 1.92 1.99 2.02 1.98 1.95 1.86 1.81 1.75 1.75 1.74 1.80 2.00 1.77 1.89 2.05 2.22 2.32 2.61 2.90 3.10 3.29 3.55 3.87 3.57 3.73 3.95 4.08 4.19 4.46 4.63 4.71 4.78 4.63 4.25 4.09 3.15 2.99 2.63 2.74 2.78 2.55 2.55 2nd 3.10 3.04 3.50 3.62 4.00 4.77 5.01 5.33 5.32 5.49 5.86 6.16 6.67 6.82 6.92 6.55 6.35 6.10 6.05 6.02 6.16 6.82 5.49 5.56 5.74 5.89 5.72 6.03 6.31 6.34 6.35 6.36 6.25 6.51 6.83 7.24 7.50 7.78 8.39 8.75 8.83 8.96 8.70 8.04 7.82 6.06 5.77 5.11 5.26 5.32 4.89 4.88 Mrig Bahar 3rd 4th 2.37 3.62 2.80 4.65 3.82 6.46 4.85 8.16 6.11 9.99 8.30 13.11 9.95 15.39 11.66 17.75 12.51 19.19 13.97 20.94 15.42 23.27 17.14 25.83 18.53 27.93 19.35 28.56 19.92 29.24 18.75 27.64 18.07 26.64 17.30 25.43 17.12 25.14 17.09 24.93 17.44 25.44 19.23 28.05 15.98 23.86 16.06 24.47 16.49 25.32 16.77 26.10 16.45 26.03 17.60 28.00 18.57 29.73 18.54 30.19 18.91 30.56 19.30 31.40 20.13 32.08 20.64 33.11 20.96 32.88 22.12 34.80 22.90 35.94 23.53 36.87 25.05 39.23 26.00 40.72 26.24 41.04 26.47 41.41 25.61 40.28 23.44 37.05 22.57 35.44 17.34 26.93 16.49 25.45 14.49 22.30 14.88 22.81 14.94 22.93 13.64 20.81 13.57 20.61 5th 4.06 5.68 7.98 10.06 12.31 15.69 18.36 20.80 21.96 23.97 25.79 28.30 30.52 31.97 32.75 30.92 29.82 28.49 28.20 27.98 28.58 31.54 26.54 27.40 28.55 29.59 30.02 32.62 35.34 36.38 37.54 39.17 40.16 38.58 40.30 42.55 43.86 44.86 47.69 49.46 50.99 51.30 49.49 45.26 43.49 33.46 31.60 27.69 28.48 28.72 26.11 25.98 M W 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 1st 1.67 1.69 1.73 1.67 1.69 1.73 1.80 1.86 1.82 1.80 1.73 1.69 1.64 1.65 1.65 1.71 1.91 1.68 1.80 1.93 2.07 2.15 2.42 2.68 2.86 3.04 3.29 3.59 3.91 4.14 4.42 4.60 4.77 4.35 4.52 4.55 4.59 4.43 4.04 3.88 3.00 2.86 2.52 2.59 2.61 2.38 2.36 2.37 2.21 2.41 2.41 2.53 Hasta Bahar 2nd 3rd 4th 3.47 2.65 4.05 3.71 3.42 5.68 3.98 4.34 7.34 3.97 5.31 8.93 4.16 6.36 10.41 4.46 7.76 12.27 4.80 9.53 14.74 5.10 11.15 16.98 5.20 12.22 18.75 5.28 13.43 20.13 5.28 13.91 20.99 5.24 14.60 22.00 5.41 15.02 22.64 5.66 16.06 23.70 5.87 16.90 24.81 6.03 17.25 25.44 6.69 19.03 28.05 5.87 16.63 24.44 6.21 17.57 25.81 6.67 18.95 27.64 7.10 20.13 29.36 7.32 20.65 30.12 7.50 21.85 32.62 7.88 22.75 34.66 7.98 22.94 35.21 8.08 22.99 35.78 8.13 23.38 37.01 8.29 24.21 38.50 8.50 25.02 40.05 8.46 24.75 40.29 8.52 25.36 41.00 8.23 25.00 40.68 7.71 24.82 39.57 7.94 25.18 40.39 8.27 25.38 39.82 8.36 25.53 40.17 8.43 25.77 40.44 8.23 24.88 38.99 7.59 22.68 35.52 7.33 21.78 34.12 5.63 16.74 26.18 5.36 15.84 24.78 4.73 13.92 21.90 4.90 14.30 22.60 4.99 14.40 22.61 4.58 13.10 20.35 4.56 13.04 20.12 4.59 13.01 20.02 4.23 11.98 18.38 4.63 12.98 19.92 4.62 12.87 19.63 4.85 13.49 20.49 5th 4.55 6.93 9.07 11.01 12.82 14.68 17.58 19.89 21.45 23.05 23.26 24.10 24.74 26.53 27.79 28.45 31.40 27.39 28.94 31.02 32.98 33.87 36.28 38.83 39.71 40.56 42.68 44.86 47.60 48.55 50.36 50.74 49.54 47.07 48.81 49.11 49.36 47.45 43.19 41.44 32.52 30.69 26.90 27.61 27.75 25.28 24.98 24.86 22.94 24.95 24.63 25.83

Journal

Journal of Applied Horticulture, 14(1): 51-55, 2012

Appl

Effect of different mulch materials on the incidence and severity of okra mosaic virus (OMV) in okra
K.T. Kareem*, O.O. Alamu, R.K. Egberongbe and O. Arogundade National Horticultural Research Institute, PMB 5432, Idi-Ishin, Jericho, Ibadan, Oyo State, Nigeria. *E-mail: rabkareem2@yahoo.com

Abstract
The study was conducted from June to September, 2010 to assess the impact of different mulch materials on the incidence and severity of okra mosaic virus (OMV) in okra cv. LD 88-1 in Ibadan, Nigeria. The overall effects of the different mulches were assessed on the incidence and severity of OMV and the resultant effect on the number of pods and pod biomass. The mulches assessed in the eld experiment were Azadirachta indica (neem) leaves, Eugenia uniora (pitanga) leaves, Terminalia catappa (tropical almond) leaves, Panicum clippings and black plastic polythene. Positive and negative controls included hoe-weeded and unweeded plots, respectively. Results indicated that at 5 weeks after sowing (WAS), there was no signicant difference in the OMV incidence on plants mulched with A. indica, E. uniora and T. catappa with values ranging from 11.91 to 15.48% while a low virus incidence of 0.5% was recorded for the plastic mulched plants. The mean virus disease severity ranged from 0.7 to 4.0 on a scale of 1-4 scoring system with plastic mulched plants showing little or no symptom of OMV at 5 WAS. However, the plants on the unweeded plots were stunted with deformed fruits. Similar trend was observed at 7 WAS with plastic mulched plot having the least incidence and severity score while the unweeded plot has the highest OMV incidence and severity. Of all the mulch materials, plots mulched with Panicum produced the least yield values while plastic mulch induced the highest yield on the okra plants. Comparing the mean number of pods of weeded and unweeded control plots; the weeded plot produced average value of 23.00.1 pods/plant while the unweeded plot produced average of 12.00.15 pods/plant. The results obtained showed that mulches especially plastic are effective in controling okra mosaic virus. Key words: Okra, okra mosaic virus, mulches, disease severity, Azadirachta indica, Eugenia uniora, Terminalia catappa, Panicum, black plastic polythene, fruit yield

Introduction
Okra (Abelmoschus esculentus (L.) Moench) is a member of the family Malvaceae and is a popular vegetable of considerable value. It is widely grown in tropical and sub-tropical areas for its immature pods that are used in salads, soups and stews (Ndunguru and Rajabu, 2004). It is among the most commonly cultivated vegetables throughout Nigeria and other tropical regions because of its much liked mucilaginous or draw property of the fruit and its ability to grow well under most tropical conditions (Vincent et al., 2005). It is a good source of calcium derived from fruits (90 mg/100 g) and leaves (70 mg/100 g). Its secondary use is the production of oil which is 20% content of the seed. Amino acids found in A. esculentus seeds compare favourably with those in poultry eggs and soybean (Hamon and Charrier, 1997). Okra is susceptible to at least 19 plant viruses with Okra mosaic virus (OMV) and okra leaf curl virus (OLCV) being the most common and well studied (Swanson and Harrison, 1993). OMV has been reported from Cote d Ivoire, Kenya, Nigeria and Sierra Leone in Africa (Brunt et al., 1996). OMV is transmitted in a non-persistent manner by the coleoptera Podagrica decolorata (Brunt et al., 1990). The use of mulch materials in the reduction of mosaic virus diseases in plants have been reported in literature. Reective plastic mulch delayed and reduced the severity of silverleaf whitey infestations in zucchini squash, pumpkins and cucumber (Summers and Stapleton, 2002). The onset of virus disease symptoms was delayed by 3 to 6 weeks in plants grown

with plastic mulch, which was critical for normal owering and fruiting (Stapleton and Summers, 2002). In zucchini squash grown with straw mulch, yields were as high and the incidence of aphidborne virus diseases was no greater than in plants grown over reective plastic mulch (Summers et al., 2004). Surface mulching either by synthetic plastic sheets or natural organic waste material offers protection for plants against root borne diseases, in addition to moisture conservation, temperature amelioration and weed control. Organic mulches including sawdust, dry grass (lawn clippings), maize cobs, rice and wheat straw, water hyacinth etc., have been very effective for vegetable growth and yield by improving moisture content of soil, heat energy and add some of the organic nitrogen and other mineral to improve nutrient status of the soil (Saeed and Ahmad, 2009). Mulching has been used to obtain good vegetable growth and yield in crops like sweet potato, potato, tomato and pepper (Awodoyin and Ogunyemi, 2005; Rahman et al., 2006). This study was conducted to assess the effect of different mulch materials on the incidence and severity of okra mosaic virus and the overall effect on the yield and biomass of okra pods.

Materials and methods

Field experiment was conducted in June 2010 through September 2010 under rain fed condition, at one of the experimental elds of National Horticultural Research Institute, Idi-Ishin, Ibadan (Latitude 7o 54N, and Longitude 3o 54E, 213 meters above

52

Effect of different mulch materials on okra mosaic virus incidence and severity

the sea level), Nigeria. Ibadan is in the rainforest-savanna transition ecosystem of south-west Nigeria. Okra seeds of LD 88-1 variety was obtained from NIHORT, Ibadan, which is a very popular and early maturing variety grown in Nigeria. There were ve mulch treatments and two control treatments. The mulch treatments included Terminalia catappa (Tropical almond) leaves, Azadirachta indica (Neem) leaves, Eugenia uniora (Pitanga) leaves, Panicum clippings (obtained from NIHORT elds) and black polythene sheet (0.25 m thick) used as plastic mulch sourced from polythene distributor in Ibadan. Each mulch material was applied at 10 t/ha, which gave 9 kg mulch material per plot of 3 x 3 m. Positive and negative controls included plots that were weeded by hoeing twice and unweeded plots, respectively. Okra variety LD 88-1 was sown on 12th June, 2010. Two seeds were planted per hole and the emerged seedlings were later thinned to one at a spacing of 60 x 30 cm to give a plant population of 55, 556 plants/ha and each plot had 28 plants. Randomized complete block design (RCBD) was used with three replications. Disease incidence: All the plots were randomly sampled in each treatment for distinct virus symptoms such as mosaic, leaf mottle, curling and malformation, plant stunting, and pod abnormalities. Disease incidence was estimated by counting the number of symptomatic plants and expressed as a percentage of the total plants sampled. Recording of disease incidence in the experimental plots was carried out 5 and 7 weeks after sowing (WAS) and the incidence of okra mosaic virus disease was recorded by visual diagnosis method. The visible symptoms of the disease were critically observed and the infected plants were identied according to Givord et al. (1972).
Number of symptomatic plant(s) Disease incidence (%) = x 100 Total number of sampled plants

Results
Disease incidence and severity of OMV: At 5 weeks after sowing (WAS), okra mosaic virus disease severity score ranged from 0.70 to 4.00 with plants in plastic mulched plot showing little or no symptoms of OMV while the plants on the unweeded plot had the highest score of 4.00 with plants having stunted stems, chlorotic leaves and deformed pods. Similar trend was observed at 7 WAS with the plastic mulched plot recording a disease severity score of 0.80 while the unweeded plot recorded a severity score of 4.0 (Table 1). The disease severity score of plants mulched with A. indica, E. uniora, T. catappa and the hoe-weeded plot at 5 WAS gave mean values of 1.67, 1.90, 1.33 and 2.67 respectively. However, the average disease severity score at 7 WAS for A. indica, E. uniora, T. catappa and the hoe-weeded plot were 1.70, 1.95, 1.38 and 2.70, respectively. The mean severity score of Panicum mulched plots at 5 and 7 WAS were 3.33 and 3.50 respectively.
Table 1. Disease incidence and severity of Okra mosaic virus as affected by different mulch materials Treatments 5 WAS 7 WAS Incidence (%) Severity Incidence (%) Severity 11.91c 1.67d 13.52d 1.70de A. indica 15.48c 1.90d 15.80d 1.95d E. uniora 13.09c 1.33de 14.90d 1.38de T. catappa 50.09b 3.33b 56.60b 3.50b Panicum Plastic 0.50d 0.70e 3.57e 0.80e Hoe-weeded 19.05c 2.67c 20.00c 2.70c Unweeded 64.64a 4.00a 70.70a 4.00a Means followed by the same letter along the column are not signicantly different according to Duncan multiple range test (DMRT) (P=0.05).

Disease severity: Disease severity was assessed using a 1-4 scoring system, (0: symptomless), 1: mild mosaic present, no leaf distortion; 2: slight distortion, chlorosis affecting <ca. 40% leaf area; 3: severe mosaic/leaf distortion, chlorosis affecting ca. 80% leaf area; 4: leaves severely distorted and stunted, 80-100% leaf area chlorotic (Ndunguru and Rajabu, 2004) and this was carried out at 5 and 7 WAS. Measurement of pod biomass and fruit yield: Number of pods per plant was determined by counting the pods produced by the okra plants. The fresh weight of pods was obtained by weighing the fresh pods on a sensitive weighing balance while for the dry weight, pods were sun dried until constant weights were obtained; later the pods were weighed. Weed species composition: Weeds were destructively sampled in each plot at 7 WAS using 0.5 m2 quadrat and weed samples were separated into broad leaves and grasses. They were identied to species level and the names were conrmed using Akobundu and Agyakwa (1998). Data analysis: The data were analysed using analysis of variance (ANOVA) by SPSS (version 16.0) statistical computer software. Duncan multiple range test (DMRT) was used to test for level of signicance at 5% probability level.

The result on the incidence of okra mosaic virus with respect to the different mulch materials indicated that all the plants in the plots showed varying degree of OMV symptoms. The negative control plot (unweeded) had the highest incidence of 64.64% followed by the plot mulched with Panicum (50.09%) at 5 WAS. At 7 WAS, 70.7% incidence was recorded for the unweeded plot while 56.6% virus incidence was obtained from the Panicum mulched plot (Table 1). At 5 WAS, the incidence of the positive control plot (hoe-weeded) was not signicantly different from those plots mulched with A. indica, E. uniora and T. catappa with percentage incidence values ranging between 11.91 and 19.05%. Similarly, there was no statistical difference in the percent incidence of plots mulched with A. indica, E. uniora and T. catappa but the incidence of these three mulch materials differed signicantly from those of the hoe-weeded plot. The virus incidence of the plastic mulched plot was 0.5% at 5 WAS and 3.57% at 7 WAS (Table 1). Effect on pod biomass and fruit yield: The least number of pods was recorded on the unweeded plot while the highest was recorded on the plastic mulched plot. The average number of pods of the weeded plot (23.00.10) was higher than that of the plot mulched with Panicum (19.00.07). The mean number of pods recorded on plots mulched with A. indica, E. uniora, T. catappa and plastic were 24.00.13, 25.00.02, 27.00.05 and 30.00.03, respectively (Table 2). Results of the pod biomass showed that the unweeded plot had the least values of 146.00.61 g and 45.00.15 g for both fresh and dry weights of pods per plant, respectively while the weeded plot produced 253.00.10

Effect of different mulch materials on okra mosaic virus incidence and severity and 105.00.28 g, respectively (Table 2). The use of plastic mulch gave the highest pods fresh weight (378.0 0.44 g/plant) while the use of Panicum mulch resulted in pods with lesser fresh weight value of 1620.54 g/plant. A. indica, E. uniora and T. catappa mulched plots produced pods with fresh weight values of 2400.35 g, 2880.12 g and 2840.15 g, respectively (Table 2). The trend of the result obtained for dry weights of pods was similar to what was observed in the fresh weights. The plants on the Panicum mulched plot produced pods with the least dry weight (760.08 g) while plants on the plastic mulched plot had pods with the highest dry weight values (233.00.29 g). The performance of E. uniora, was better than that of T. catappa while that of T. catappa was higher than that of A. indica (Table 2). Generally, of all the mulch materials, plastic mulched plot produced the highest yield components while Panicum mulched plot produced the least yield components. Nonetheless, the yield produced by plots mulched with Panicum was greater than that produced by the unweeded plots but not as much as that produced by the weeded plots. Effect of mulch materials on weed species composition: A total of 20 weed species were recorded across the mulched plots, 14 were broad leaves while 6 were grasses (Table 3). All the mulched plots have varying degrees of weed ora composition. The plastic mulched plot had the least number of weeds. The broad leafed weed species found in the plastic mulched plot were Calopogonium mucunoides and Oldenlanda herbacea while the other plots had at least 3 species of broad leafed weeds. Tridax procumbens was the most abundant broad leafed weed species in all the plots but the plastic mulched plot was able to resist this weed. The plastic mulched plot could not resist the growth of the grasses as much as it did for the broad leaves. T. catappa mulched plot had the least number of grasses followed by Panicum mulched plot. Cyperus rotundus and P. maximum were found in all plots except the T. catappa mulched plot.

53

Table 2. Effect of the different mulch materials on yield and biomass components of okra plants Mulch No of Pods/plant Fresh weight of materials Pods/plant (g) 24.0 0.13 240.0 0.35 A. indica 25.0 0.02 288.0 0.12 E. uniora 27.0 0.05 284.0 0.15 T. catappa 19.0 0.07 162.0 0.54 Panicum Plastic 30.0 0.03 378.0 0.44 Hoe-weeded 23.0 0.10 253.0 0.10 Unweeded 12.0 0.15 146.0 0.61 Each value is a mean standard error Dry weight of Pods/plant (g) 125.0 0.18 162.0 0.21 145.0 0.06 76.0 0.08 233.0 0.29 105.0 0.28 45.0 0.15

Discussion
The results obtained in this investigation showed that all the mulch materials except Panicum improved the performance of okra plants compared to the unmulched plants. Hudu et al. (2002) and Awodoyin and Ogunyemi (2005) also reported the superiority of mulched plants over unmulched plants. Very few of the plastic mulched okra plants were infected with OMV while the incidence was high in the unweeded plot. There have been several reports on plastic mulch reducing the incidence of mosaic diseases in vegetables. Vani et al. (1989) stated that yellow polythene mulch reduced the incidence of mosaic disease on muskmelons. Moreso, Lutzinsky et al. (1996) demonstrated that leaf curl incidence in tomato was low in rows mulched with yellow or brown plastic. The highest severity of OMV in the unweeded plot may be explained by the fact that weeds serve as reservoirs of viruses, thus bringing about increased incidence and severity. Insects can transmit the viruses to the okra plants during non-persistent feeding, transmission can be through mechanical transmission or through other means. It has been reported that volunteer plants and weeds provide shelter and sources of nutrients for

Table 3. Weed species and their frequencies as inuenced by the different mulch materials Weed species Mulch material Plastic T. catappa A. indica E. uniora A. Broad leave weeds Acalypha mbriata Schum. & Thonn Ageratum conyzoides L. Amaranthus spinosus L. Amaranthus viridis L. Calopogonium mucunoides Desv. Celosia leptostachya Benth. Euphorbia heterophylla L. Mitracarpus villosus (Sw.) DC. Oldenlandia corymbosa L. Oldenlandia herbacea (L.) Roxb. Spigelia anthelmia L. Stylosanthes gracillis L. Tridax procumbens L. Talinum fruticosum (L.) Juss. B. Grasses Axonopus compressus (Sw.) P. Beauv. Commelina benghalensis L. Cyperus rotundus L. Panicum maximum Jacq. Paspalum conjugatum Berg. Paspalum scrobiculatum L. 1 0 0 0 4 3 0 0 0 0 0 0 6 0 1 1 0 0 0 2 0 1 0 3 0 2 0 0 0 0 0 0 12 0 6 0 2 1 0 0 0 0 0 0 1 2 1 1 0 3 0 0 3 0 0 4 4 3 4 0 0 0 0 0 2 0 0 0 0 1 0 0 0 0 1 1 1 1 1 1

Panicum 0 0 2 0 0 3 4 0 8 0 0 0 10 1 0 0 2 4 0 0

Hoe-weeded 0 0 1 0 0 1 2 0 1 4 1 2 12 0 0 1 2 4 0 0

Unweeded 0 0 0 2 4 0 5 0 0 0 0 0 10 0 0 3 2 4 0 4

54

Effect of different mulch materials on okra mosaic virus incidence and severity in cayenne pepper (Awodoyin and Ogunyemi, 2005). The yields obtained from the mulched plots were higher than that of the hoe-weeded plot; the only exception was the Panicum mulched plot which had values below that of the hoe-weeded plot. Despite this fact, the use of Panicum as mulching material should be given consideration because weeding of agricultural land is expensive and Panicum mulched plot gave higher yield than un weeded plot. Chianu and Akintola (2003) stated that weed control requires more labours which limits the land area a farmer could cultivate. Usoroh (1983) reported that weeding alone takes 30-45% of total cost of labour required for fruit and vegetable production in Nigeria. In conclusion, mulching reduces incidence and severity of OMV and enhances the yield of okra plants by reducing weed infestation and improving the crop growing environment. This helps to reduce the cost incurred on weeding and increase agricultural productivity. Growers should be encouraged to adopt the use of mulches for growing okra in areas where okra mosaic virus is a problem.

virus vectors (Akel et al., 2010). Other vegetative structures or contaminated weed seeds may also harbor viruses. Apart from acting as alternative source of inoculum, these plants sustain the viability of the virus between crop seasons (Duffus, 1971). Furthermore, wild plants growing within the same elds as crops or in nearby areas can be inoculum sources and reservoirs of viruses for crops. The relationship between crops and disease organisms is generally a complex annual cycle involving crops, vectors and weeds. The disease severity of Panicum was equally high. This result is not surprising since Panicum itself is a weed and may serve as a reservoir of viruses. It has been reported that weeds are known to play important roles in the spread and epidemiology of virus diseases (Dufus, 1971; Sidek et al., 1993). Many insect and nematode pests, which infect crops with viruses causing major diseases, have often been shown to acquire the viruses initially from weeds (Zimdahl, 1980). Moreso, Awodoyin et al. (2007) reported that grasses do not provide long cover over the soil because of rapid decomposition. Therefore, the rapid decomposition might enhance increase in the population of microorganisms including viruses and encourage rapid weed reinfestation and this may increase transmission of viral diseases. The study revealed that mulching materials increased the yield of okra plant by increasing the number of pods, with plastic mulched plot having the highest yield followed by A. indica, E. uniora and T. catappa mulched plots in decreasing order. This statement agrees with the previous studies which revealed that besides weed control, many advantages of using black polythene justify its use: better water use efciency, higher yields and better quality, earlier plant development due to increased soil temperature, etc. (Scott 2005; Ghosh et al. 2006). The yields of A. indica, E. uniora and T. catappa mulched plots were higher than that of the Panicum mulched plots. This may be due to the fact that the other three mulch materials are broad leaves and do not decompose rapidly as compared to Panicum which has narrow leaves and decomposes faster. Awodoyin et al. (2007) also stated that grass does not provide long cover over the soil because of rapid decomposition. Nonetheless, the yield of Panicum mulched plot was higher than that obtained from the unweeded plot. Ramalan and Nwokeocha (2000) reported that the effect of rice straw on crop yield was found to be variable but was normally higher than bare soil. However, in most of the publications the general trend is that straw mulch has positive effects on the crop, soil moisture, etc., but achieves only an intermediate yield improvement compared to black plastic mulch (Alcantara et al., 2007; Cirujeda et al., 2007). In addition, Summers et al. (2005) reported that plants grown over straw mulch produced higher overall yields, including large size melons, than those grown over bare soil. All mulched plots had high fresh and dry pod weights with plastic mulched plot having the highest value. However, the unweeded plot had the least pod biomass value. Mulching is an effective method of manipulating crop growing environment to increase yield and improve product quality by controlling weed growth, ameliorating soil temperature, conserving soil moisture, reducing soil erosion, improving soil structure and enhancing organic matter content (Hochmuth et al., 2001; Awodoyin and Ogunyemi, 2005). Moreso, weeds reduce crop productivity by interfering with crop growth. In Nigeria, uncontrolled weed reduced yield by about 40% in maize and 84% in upland rice (Akobundu, 1980), 31-70% in groundnut (Lagoke et al., 1981) and 73-78%

Acknowledgements
We thank the NIHORT for research facilities and to Dr Awodoyin, R.O. of University of Ibadan, Department of Crop Protection for his constructive criticism of this manuscript.

References
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55

Rahman, M.J., M.S. Uddin, S.A. Bagum, A.T.M.A.I. Mondol and M.M. Zaman, 2006. Effect of mulches on the growth and yield of tomato in the costal area of Bangladesh under rain fed condition. Int. J. Sustain. Crop. Prod., 1: 6-10. Ramalan, A. and C. Nwokeocha, 2000. Effects of furrow irrigation methods, mulching and soil water suction on the growth, yield and water use efciency of tomato in the Nigerian Savanna. Agric. Water Manag., 45: 317-330. Saeed, R. and R. Ahmad, 2009. Vegetative growth and yield of tomato as affected by the application of organic mulch and gypsum under saline rhizosphere. Pak. J. Bot., 41(6): 3093-3105. Scott, G. 2005. Biodegradable plastics in agriculture. In: Biodegradable Polymers for Industrial Applications. R. Smith Woodhead (Ed.) London, UK. pp. 1-20. Sidek, Z., H. AbdulSamad and I. Sulaiman, 1993. Detection of cucumber mosaic virus in weeds in Malaysia using dot immunobinding assay. J. Pl. Prot. Trop., 10(2): 111-117. Stapleton, J.J., C.G. Summers, 2002. Reective mulches for management of aphids and aphid-borne virus diseases in late-season cantaloupe (Cucumis melo L. var. cantalupensis). Crop Protec., 21: 891-8. Summers, C.G., and J.J. Stapleton, 2002. Use of UV reective mulch to delay the colonization and reduce the severity of Bemisia argentifolii (Homoptera: Aleyrodidae) infestations in cucurbits. Crop Protec., 21: 921-8. Summers, C.G., J.P. Mitchell and J.J. Stapleton, 2004. Non-chemical insect and disease management in cucurbit production systems. Acta Hort., 638: 119-25. Summers, C.G., J.P. Mitchell and J.J. Stapleton, 2005. Mulches reduce aphid-borne viruses and whiteflies in cantaloupe. Carlifornia Agriculture, 59(2): 90-94. Swanson, M.M. and B.D. Harrison, 1993. Serological relationships and epitope proles of an isolate of okra leaf curl geminivirus from Africa and the Middle East. Biochemistry, 75: 707-711. Thottappilly, G. 1992. Plant virus diseases of importance to African agriculture. J. Phytopathol., 134: 265-288. Usoroh, N.J. 1983. Weed problems and control strategies in fruit and vegetable production in Nigeria. Acta Horticulturae, 123: 299304. Vani, S., A. Varma, T.A. More and K.P. Srivastava, 1989. Use of mulches for the management of mosaic disease in muslmelon. India. Phytopathol., 42: 227-235. Vincent, B.O., T.M. Michael and M.M. Stanley, 2005. Effect of cattle manure application on yield and yield indices of okra (Abelmoschus esculentus L. Moench). Journal Food Agriculture Environment, 3: 125-129. Zimdahl, R.L. 1980. Weed-Crop Competition A Review. Intl. Plant Protection Center, Oregon State Univ. Corvallis. 196pp.
Received: July, 2011; Revised: August, 2011; Accepted: January, 2012

Journal

Journal of Applied Horticulture, 14(1): 56-62, 2012

Appl

Effect of putrescine, GA3, 2, 4-D, and calcium on delaying peel senescence and extending harvest season of navel orange
H.A. Kassem*, H.A. Marzouk1 and R.S. Al-Obeed
1

Department of Plant Production, College of Food and Agricultural sciences, King Saud University, Saudi Arabia., Department of Pomology, Faculty of Agriculture, Alexandria University, Alexandria, Egypt, *E- mail: hkassem60@yahoo.com

Abstract
The present study was conducted in 2007/2008 and 2008/2009 seasons in order to extend harvest season and maintain fruit quality for better marketability of Washington navel oranges growing in clay soil by preharvest foliar sprays of GA3, 2,4-D, putrescine and calcium either alone or in combinations. Fruits were harvested on two different harvest dates, the rst was at the estimated commercial harvest date (middle December), and the second was late in the harvest season (during February). At both harvesting dates, all spray treatments delayed fruit softening, peel ageing and fruit color break and decreased creasing and fruit drop. Also, fruit TSS, sugars and vitamin C contents increased. The treatments had positive inuence on extending harvest season without any deterioration in fruit characteristics. Spraying the different substances in combinations gave better results, especially with putrescine. Key words: Putrescine, GA3, 2, 4-D, calcium, peel senescence, Navel orange

Introduction
Oranges are one of the worldwide popular fruits mostly consumed fresh. Orange fruit faces, pre- or post-harvest, a number of rind disorders such as creasing, splitting, pufng, peel pitting and senescence. Delaying peel senescence prolongs the fruits life, improves fruit quality and extends its marketing season, which enhances the crop value and contributes to growers returns. Thus, any manipulation of the very nal stage of fruit development in order to delay rind senescence improves fruit quality and extends its exporting season. In Egypt, navel orange growers tend to expand harvest period by keeping the fruit for longer time on the trees to extend marketing season, for late export date. This practice leads to the appearance of the previous mentioned disorders, mainly on the rind, ending with fruit senescence and shorten its shelf life and marketability. Controlling of rind aging, mainly rind softening in navel oranges is important for the marketing of quality fresh orange and to prolong the life of the fruit with high quality characters as long as possible after harvest. Growth regulators are one of several tools, when properly used, enable citrus growers to extend marketing period with no loss of fruit quality (Ismail, 1997). Gibberellic acid when applied pre-harvest retarded rind softening and fruit maturation (Ismail 1997; Coggins, 1981). Creasing, splitting, puffing and peel pitting can be reduced in intensity or minimized using gibberellic acid and synthetic auxins or a mixture of both (Agusti et al., 2002). According to Chapman (1983), exogenous applications of GA3 at the citrus fruit color change stage maintained the peel quality of late-harvested fruit and reduced mesocarp cracking. In addition, pre-harvest sprays of 2,4-D alone or with GA3 is proven effective technique for better peel quality in tree-stored fruit, and reducing late season fruit drop therefore, extending the harvest

season, as well as retarding rind senescence and lowering fruit decay (Golddchmidt and Eilati, 1970; Ismail, 1997). Moreover, Tumminelli et al . (2005) noticed an increase in ethylene production in the albedo tissues of Satsuma mandarin with the ripening stage and it increased with peel aging. Zheng and Zhang (2004) reported a gradual decline in the concentrations of free polyamines in mandarin fruits after harvest that was parallel to peel senescence. Polyamines are group of natural compounds that are believed to have anti-senescence function by inhibition of the formation of enzymes essential to the synthesis of ethylene (Ke and Romani, 1988) thus, retard ripening and extend fruit shelf life. They also improve fruit quality by reducing mechanical damage and increasing fruit rmness (Valero et al., 1998a and b, PerezVicente et al., 2002). In addition, many researches focused on extending citrus fruit life by pre- or post-harvest treatments with calcium (El-Hilali et al., 2004; Valero et al., 1998b). In view of the above ndings, the present study was conducted to evaluate the effect of pre-harvest foliar sprays of GA3, 2, 4-D, putrescine and calcium on internal and external quality of navel orange fruit and fruit abscission during late in the season, to determine whether it is possible to prolong the shelf life and delay the harvest of navel orange trees growing in clay soil without economical loss.

Materials and methods

Plant material and treatment: Thirty-ve years old Washington navel orange trees (Citrus sinensis, L.), budded on sour orange rootstock planted at 4.5x4.5 m apart in a private citrus orchard at El-Tarh region, EL- Behera Governorate were selected for the study. The soil was clay, well-drained with water table about 110 cm and pH 8. Trees were subjected to the standard cultural practices in the orchard. In January of both seasons, calcium superphosphate (15.5% P2O5) was added at the rate of 250 kg

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange per feddan. Ammonium nitrate (33% N) was applied at the rate of 250 kg in March, 250 kg in May and 200 kg in August of both seasons per feddan. In August of both seasons, 100 kg per feddan potassium sulfate (48% K2O5) was added. Trees were irrigated with Nile water every 15-20 days. One hundred and ten trees were selected as uniform as possible and were subjected to two foliar sprays. The rst was at the beginning of fruit color change (about month and half before normal harvest date), and the second after 25 day from the rst spraying date. Trees were sprayed with, GA3 (10 ppm), 2, 4-D (10 ppm), putrescine (PUT) (5 mmol) and chelated calcium (1500 ppm) either alone or in combinations. Treatments were arranged in a complete randomized design with ve replicates (each replicate consisting of two trees) and the trees were treated with eleven foliage treatments (2 x 5 x 11 = 110 trees), water only, GA3, 2,4-D, putrescine, calcium, GA3 + 2,4-D, GA3 + putrescine, GA3 + calcium, 2,4-D + putrescine, 2,4-D + calcium, and putrescine + calcium. The surfactant Nourlm (produced by Alam Chemca) at the rate of 40 cm/100 L water was added to all sprayed chemicals in order to obtain best results. Trees were harvested at two different dates, the rst at the estimated commercial harvest date (middle September), and the second late in the harvest season (during February). Determination of fruit physical characteristics: The percentage of pre-harvest fruit drop, rind ageing, softening, creasing and fruits unt for export and peel thickness (mm) and fruit color were recorded at each harvest date (middle September and February). Peel softening and fruits unt for export were measured depending on the scale of export specications. Rind ageing was estimated as the percent of fruit with peel pitting. Fruit color was estimated by giving ve degree of color stage as follows; 1= 100 % green, 2= 25% green, 3= 50% orange, 4= 75% orange and 5=100% orange. Determination of fruit chemical characteristics: A sample of ten fruits were taken from each replicate at each harvest date in both growing seasons in order to estimate electrolyte leakage (EL), fruit carotenoids, total soluble solids (TSS), acidity, TSS/ acidity ratio, sugars, and vitamin C (VC) contents. Acidity (%) and VC (mg/100 mL juice) was determined by titration according to AOAC (1980). Electrolyte leakage (EL) as ppm was estimated in fruit peel by Conductivity/TDS Meter. Carotenoids (mg/100 g peel fresh weight) were measured according to the method of Moran and Porath (1980). Sugar content (%) was determined according to the method of Malik and Singh (1980). Statistical analysis: Data obtained were subjected to analysis of variance (ANOVA) to detect treatment effect. Mean separation were performed and compared using least signicant difference (LSD) at P 0.05 level. The data were analyzed using Statistical Analysis System (SAS) version 6.03.

57

alone, 2,4-D alone in both seasons, and PUT + 2,4-D in the rst season) increased fruit peel thickness when compared with the control in both seasons. Spraying GA3 either alone or + PUT, 2, 4-D or Ca had signicanty higher effect in increasing peel thickness than 2,4-D + Ca in the rst season only. In addition, fruit color break was delayed signicantly by spraying PUT, GA3, PUT + 2, 4-D, PUT + GA3 and GA3 + Ca in both seasons. A signicant decrease in the rind ageing percent was recorded in both seasons by all foliar sprays (except 2, 4-D alone in the rst season). The percent of fruit softening, fruit creasing, fruit drop and fruits unt for export were decreased by all foliar sprays in both seasons as compared with the control. All foliar sprays had higher effect in decreasing fruit softening than spraying 2,4-D alone in both seasons. Also, spraying PUT alone resulted in the lowest creasing percent in the rst season when compared with 2, 4-D alone, 2, 4-D + GA3, 2, 4-D + Ca and GA3 + Ca. At the second harvest date (Table 2), peel thickness increased signicantly in the rst season by spraying GA3 alone, Ca alone, PUT + Ca and 2,4-D + Ca with no signicant differences among them. However, in the second season foliar sprays of Ca alone, PUT + 2, 4-D, PUT + GA3, 2, 4-D + GA3 and Ca + GA3 resulted in higher increase in fruit thickness in comparison with the control, with the highest increase obtained by GA3 + Ca. On the other hand, fruit color was not signicantly affected by any of the sprayed substances as compared with the control in both seasons. All treatments (except PUT + GA3 and 2, 4-D + Ca) decreased rind aging in the rst season, whereas, no signicant effect was obtained in the second season as compared with the control. The percents of rind softening and creasing were decreased by all foliar sprays in comparison with the control. In general, spraying the substances in combinations resulted in the highest decrease in rind softening and creasing. In both seasons, the percent of late in the season fruit drop and fruit unt for export was signicantly decreased by all treatments when compared with the control. The data of the present study showed that in general all sprayed growth regulators had positive inuences in decreasing fruit external characteristics disorder and delaying fruit senescence at both harvesting dates. Senescence is the nal stage of fruit growth and development. It is a process mainly characterized by disintegration of organelle structures, intensive loss of chlorophyll and proteins, membrane leakage and breakdown of cell wall components leading to loss of tissue structure (Paliyath and Droillard 1992, Buchanan-Wollaston 1997), which all contribute to the weakening of peel structure and leads to the senescence of the fruit. Delaying peel and rind senescence improves fruit quality and prolongs the life of citrus fruit and extends its value (Ismail, 1997). Numerous studies have suggested the promotive role of ethylene on the process of fruit ripening and senescence (Abeles et al., 1992; Zarembinsiki and Theologis, 1994; Lelievre et al., 1997; Dilley, 1977). In non-climacteric fruits, such as citrus, ethylene is not required for the coordination of ripening of the fruit (Giovannoni, 2001; Lelierve et al., 1997; Yang and Hoffman,1984), however, it plays an important role in the senescence process. Considerable progress has been made during the past decade in understanding the possible relationship between ethylene and fruit ripening, and association between the sprayed substances specially the polyamines (for example putrescine in our study). There is evidence of an interrelationship between

Results and discussion

Fruit physical characteristics: The effect of the different treatments on fruit physical characteristics at the rst and second harvest dates are presented in Tables 1 and 2. The data of the rst harvest date showed that all treatments (except spraying PUT

58

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange

Table 1. Effect of PUT, GA3, 2, 4-D and Ca, foliar sprays on fruit physical characteristics at the rst harvest date during 2007/2008 and 2008/ 2009 Treatment Peel thickness Color Rind ageing Softening Creasing Fruit drop Fruit unt for (mm) (%) (%) (%) (%) export (%) Season 2007/2008 water 28.8c 4.86a 2.72a 0.98a 4.94a 8.47a 18.11a PUT 30.7bc 4.34b 1.16c 0.32c 1.05d 4.98b 9.86bc 2,4-D 30.6bc 4.68a 2.13a 0.54b 2.86bc 3.34c 11.23bc GA3 34.6a 4.42b 1.20c 0.26cd 1.75cd 2.38c 7.79c Ca 32.3ab 4.69a 2.05b 0.13d 2.06cd 3.50bc 8.97bc PUT +2,4-D 30.4bc 4.36b 1.30c 0.24cd 2.03cd 3.26c 7.96c PUT + GA3 34.3a 4.43b 1.14c 0.15d 1.98cd 2.04c 6.76c PUT + Ca 32.8ab 4.40b 1.18c 0.12d 2.08cd 2.13c 7.97c 2,4-D +GA3 34.6a 4.87a 1.26c 0.21cd 2.46c 2.23c 8.43c 2,4-D + Ca 31.6b 4.67a 1.30c 0.23cd 3.05bc 3.56bc 9.76bc GA3 + Ca 34.4a 4.37b 1.31c 0.19cd 2.32c 2.89c 8.43c Season 2008/2009 water 30.6b 5.00a 1.72a 2.78a PUT 32.6b 4.68ab 0.42c 0.19e 2,4-D 32.4b 4.89ab 1.13b 1.34b GA3 34.4a 4.44b 0.50c 0.78c Ca 33.8a 4.82a 0.98bc 0.53de PUT +2,4-D 33.8a 4.58b 0.60bc 0.64c PUT + GA3 34.4a 4.46b 0.44c 0.85c PUT + Ca 34.8a 4.45b 0.78bc 0.42de 2,4-D +GA3 33.9a 4.76ab 0.56c 0.51de 2,4-D + Ca 33.8a 4.96a 1.02b 0.53de GA3 + Ca 34.6a 4.64b 0.71bc 0.49de Values within a column with same letter are not signicantly different (P<0.05). Treatment Peel thickness (mm) 30.4b 32.7ab 31.8b 34.2a 35.3a 32.6ab 33.3ab 34.8a 32.7ab 35.4a 33.2ab Color Rind ageing Softening (%) (%) Season 2007/2008 8.66a 31.54a 5.26b 18.46b 4.21c 15.86c 5.26b 14.85c 3.45c 19.64b 4.78bc 6.28e 6.16ab 8.64de 5.54b 7.87e 2.28c 7.75e 10.00a 6.87e 4.23c 6.07e 7.98a 2.65bc 2.64bc 1.86bc 2.26bc 3.09bc 2.03bc 2.43bc 3.14bc 3.09bc 1.69c 10.65a 6.67bc 2.98cd 5.40bc 2.03d 3.98c 1.02d 2.94cd 1.00d 2.86cd 1.46d 22.57a 11.86bc 9.36c 10.05bc 6.87c 7.64c 6.87c 7.98c 6.87c 6.76c 5.87c

Table 2. Effect of PUT, GA3, 2, 4-D and Ca foliar sprays on fruit physical characteristics at the second harvest date Creasing (%) 28.62a 14.65b 15.34b 15.76b 17.76b 9.76cd 10.78cd 9.87cd 8.87d 10.76cd 9.87cd 37.64a 20.32c 21.14b 15.43d 20.16c 16.49d 10.63f 9.08f 13.32e 13.74e 16.06d Fruit drop (%) 18.05a 10.42b 5.37c 6.39c 5.45c 7.68c 10.60b 9.03bc 4.57c 8.64c 4.46c 20.53a 16.18b 9.57b 11.00b 10.63b 6.63c 8.85bc 10.54b 6.74c 4.58c 7.67c Fruit unt for export (%) 57.36a 30.46cd 25.67de 23.54de 21.68e 27.54d 28.87d 26.32de 23.46de 36.87b 24.67de 54.35a 34.28bc 28.26c 24.34ce 30.34c 23.48e 20.34e 32.66bc 24.68ce 28.48c 35.86b

water PUT 2,4-D GA3 Ca PUT +2,4-D PUT + GA3 PUT + Ca 2,4-D +GA3 2,4-D + Ca GA3 + Ca

5.00 4.94 5.00 4.86 5.00 4.96 4.83 4.94 4.98 5.00 5.00

Season 2008/2009 water 31.8d 5.00 6.89 34.78a PUT 33.8cd 5.00 4.86 15.64c 2,4-D 33.6cd 5.00 6.57 17.08bc GA3 34.6cd 4.87 4.60 12.87d Ca 34.8c 4.98 4.26 17.64b PUT +2,4-D 35.4bc 4.98 3.98 11.98d PUT + GA3 34.8c 4.86 3.74 10.86d PUT + Ca 33.6cd 5.00 6.21 9.76d 2,4-D +GA3 34.8c 5.00 4.68 11.87d 2,4-D + Ca 34.4cd 4.96 6.64 14.73c GA3 + Ca 43.8a 5.00 7.42 10.04d Values within a column with same letter are not signicantly different (P<0.05).

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange ethylene and polyamines during fruit ripening and senescence (Pandey et al., 2000). They play an inhibitory role on ethylene production through inhibition of ACC synthesase and ACC oxidase (Apelbaum et al., 1981; Lee et al., 1997), thus delaying ethylene emission. Polyamines have been reported to reduce softening, delay senescence and reduce decay in several fruits (Saftner and Baldi, 1990; Kramer et al., 1991). Other benecial effects of polyamines application on fruit are: retarding color changes and increasing fruit rmness (Valero et al., 1999; Valero et al., 2002). Putrescine application leads to changes in cell wall stability (Messiaen et al., 1997) by inhibition of the action of polygalacturonase and pectin methyl esterase involved in softening, and also cross-link pectic substances in the cell wall, producing rigidication and increasing fruit rmness (MartinezRomero et al., 2002; Perez-Vicente et al., 2002). This might explain the increase in rmness by putrescine application obtained for navel orange in our study. Similar results were reported for lemon by Valero et al. (1998b). Moreover, GA3 sprays in the present study increased peel thickness and peel rmness and decreased fruit ageing, creasing, color change and the number of unt export fruits. Similar results were obtained for Hamlin, Valencia, navels and blood oranges and mandarins (Coggins, 1973; Greenberg et al., 1992; Davies et al., 1997; Davies et al., 1999; Pozo et al., 2000). GA3 is known to delay and retard chlorophyll degradation in citrus (El-Otmani and Coggins, 1991; Agusti et al., 1981). Moreover, its role is not limited only to the regulation of rind color, but also in delaying the more general process of peel ageing (Baez-Sanudo et al., 1992). This might be due to the association of GA3 with the reduction of fruit peel growth as has been reported for mandarins (Pozo et al., 2000). Also, we recorded improvement in fruit physical characters by calcium sprays specially an increase in peel rmness, peel thickness and decrease in rind ageing and creasing. Sayed et al. (2004) working on grapefruit and El-Hilali et al. (2004) working on mandarin obtained similar results. Storey et al. (2005) reported fruit rind disorders as a result of calcium deciency. Calcium role in the physiological disorder related to ripening, fruit quality and shelf life is well established (Chaplin and Scott, 1980; Wimwright and Burbage, 1989). Calcium is involved in cell wall membrane metabolism and it contributes to the maintenance of conguration of specic enzymes (Jones and Lunt, 1967). Addition of calcium improves rigidity of cell walls and obstructs enzymes such as polygalacturonase from reaching their active sites (John, 1987), thereby retarding tissue softening and delaying ripening. Repeated sprays of Ca solutions increased the proportion of unaffected navel orange fruit with albedo breakdown (Treeby and Storey, 2002). The positive inuence on decreasing late in the season fruit drop by the sprayed substances in our study is obvious. It is well established that plant growth regulators are involved in controlling abscission. Ethylene accelerates mature citrus fruit abscission (Sexton and Roberts, 1982), and as previously mentioned that putrescine inhibits ethylene production, this might explain its effect on decreasing fruit drop. In addition, 2, 4-D is widely used in citrus in order to reduce the incidence of mature fruit drop and its primary action is to delay the development of the abscission layer (Coggins, 1973). GA3 and Ca sprays inuence might be due to the increase in the thickness of both juncture zone

59

and the pedical as well as increasing the connections of vascular system and cell adhesion in union zone as reported for grapefruit by Sayed et al. (2004). Fruit chemical characteristics: The data of the rst and second harvesting dates are presented in Tables 3 and 4. Data of the rst harvest date showed that all foliar sprays (except 2, 4-D alone and PUT + 2, 4-D in the rst season) increased fruit TSS content during both seasons when compared with the unsprayed control trees. Fruit acidity was signicantly decreased by all treatments (except for GA3, Ca, PUT + GA3 and GA3 + Ca) in the rst season. However, in the second season, acidity content was not signicantly affected by the treatments (except for 2, 4D alone which resulted in decreasing fruit acidity). TSS/Acidity ratio was signicantly higher than the control by spraying PUT, 2,4-D, PUT + Ca, 2, 4-D + GA3 and 2, 4-D + Ca in the rst season. In the second season, foliar sprays of GA3 alone, 2, 4-D + PUT and 2, 4-D + Ca signicantly increased TSS/acidity ratio. Vitamin C content was signicantly increased in both seasons by spraying GA3 alone, Ca alone, PUT + GA3 and GA3 + Ca with no signicant difference obtained among them. The electrolyte leakage (EL) was lower than the control by spraying PUT alone and GA3 + Ca in the rst season, whereas, in the second season all foliar sprays signicantly decreased the EL as compared with the control. The highest decrease was obtained by PUT + GA3 and PUT + Ca sprays. Fruit reducing sugars content was increased by PUT + Ca, 2,4D + GA3 and 2,4-D + Ca, whereas, it decreased due to spray of 2,4-D alone, GA3 alone, PUT + 2,4-D, PUT + GA3 and GA3 + Ca as compared with the control. Only foliar sprays of GA3 alone and 2,4-D + Ca increased reducing sugars content in the second season, whereas, it was decreased by spraying 2,4-D + GA3, PUT + GA3 + PUT and GA3 + Ca. In the rst season, fruit non reducing sugars content increased by all sprays (except PUT + GA3, PUT + Ca and GA3 + Ca). In the second season, foliar sprays of 2,4-D alone, Ca alone, 2,4-D + GA3 and 2,4-D + Ca gave higher non reducing sugars content than the control with no signicant difference among them. In both seasons, total sugars content was signicantly higher than the control by spraying GA3 alone, Ca alone and 2,4-D + Ca. A signicant decrease in fruit carotenoids content was observed as a result of spraying GA3 + Ca (in both seasons), PUT + GA3 (in the rst season) and (PUT or GA3 alone) in the second season. Data on the effect of the sprayed substances at the second harvest (Table 4) indicated that all foliar sprays (except 2, 4-D alone) increased fruit TSS content in both seasons as compared with the control. In addition, spraying of GA3 alone, PUT + GA3, PUT + CA and 2,4-D + Ca gave higher TSS content than spraying PUT alone in the rst season. A signicant increase in fruit acidity was obtained by spraying GA3 alone, Ca alone, PUT + GA3 and GA3 + Ca in the rst season. Whereas, in the second season fruit acidity was not signicantly affected by any of the treatments. The ratio of TSS to acidity increased signicantly by spraying 2, 4-D alone, 2,4-D + Ca and PUT + Ca in the rst season, whereas, in the second season all foliar sprays increased TSS/Acidity ratio when compared with the control. Vitamin C content increased in all treatments (except PUT + 2, 4-D in both seasons and 2,4-D + GA3 in the rst season) in both seasons. However, VC content was decreased by spraying 2, 4-D alone in the second season

60

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange

Table 3. Effect of putrescine (PUT), GA3, 2, 4-D and Ca foliar sprays on fruit chemical characteristics at the rst harvest date Treatment TSS EL2 Sugars (%) Acidity TSS/ VC1 (mg/100 mL ) (ppm) Reducing Non-reducing Total (%) (%) Acidity 2007/2008 Water 10.44c 1.22a 8.56b 58c 294a 3.67cd 4.27b 7.94b PUT 11.04b 1.04c 10.62a 62bc 256b 3.61d 5.20a 8.81b 2,4-D 10.64c 0.97c 10.97a 57c 268ab 3.33g 5.43a 8.76b GA3 11.08b 1.24a 8.94b 71a 280ab 3.45f 5.75a 9.20a Ca 11.44a 1.16ab 9.86ab 67ab 276ab 3.64d 5.48a 9.12a PUT +2,4-D 10.47c 1.12b 9.35b 58c 271ab 2.94h 5.63a 8.57b PUT + GA3 11.18ab 1.18a 9.47b 65b 261ab 3.21g 4.94b 8.15b PUT + Ca 11.43a 1.08b 10.58a 64b 269ab 3.84bc 4.46b 8.30b 2,4-D +GA3 11.58a 1.00c 11.58a 60c 274ab 4.94a 5.24a 10.18a 2,4-D + Ca 11.0b6 1.01c 10.95a 57c 269ab 3.98b 5.46a 9.44a GA3 + Ca 11.42a 1.26a 9.06b 67ab 249b 3.46f 4.48b 9.94a 2008/2009 Water PUT 2,4-D GA3 Ca PUT +2,4-D PUT + GA3 PUT + Ca 2,4-D +GA3 2,4-D + Ca GA3 + Ca
1

Carotenoids (mg/100g) 7.65a 6.79ab 8.03a 6.78ab 7.17ab 7.02ab 5.67b 6.23ab 7.09ab 6.98ab 5.76b 6.59ab 4.43c 7.00a 4.98bc 6.12ab 7.13a 5.89b 6.02b 7.08a 6.12ab 4.98bc

10.28d 11.42c 11.08c 11.89b 11.84b 12.36a 12.48a 11.64b 11.82b 12.48a 12.86a

1.09a 1. 00a 0.96b 1.02ab 1.10a 1.02ab 1.14a 1.06ab 1.03ab 0.98a 1.14a

9.43b 11.42ab 11.54ab 11.66a 10.76ab 12.12a 10.95ab 10.98ab 11.48ab 12.73a 11.28ab

52c 59b 50c 65a 63a 56bc 68a 56bc 59b 52c 64a

312a 280c 294b 278cd 284bc 290bc 263de 248e 266d 269cd 268cd

3.86bc 3.78cd 3.82cd 4.32a 3.82cd 4.00b 3.42de 3.77cd 3.46de 4.23a 3.36e

4.41c 4. 96bc 5.84a 4.98bc 5.22ab 4.68bc 4.86bc 4.67bc 5.22ab 5.47ab 4.86b

8.27b 8.74a 9.66a 9.30a 9.04ab 8.68b 8.28b 8.44b 8.68b 9.70a 8.22b

Vitamin C, 2 Electrolyte leakage. Values within a column with same letter are not signicantly different (P<0.05). Carotenoids (mg/100g) 8.34a 6.54b 8.78a 5.68c 6.12bc 7.12b 5.65c 6.12bc 6.56b 7.48ab 4.89c 8.97a 7.65b 8.65a 6.13bc 6.43bc 6.64bc 6.76bc 6.87bc 7.87ab 8.01ab 5.98c

Table 4. Effect of putrescine (PUT), GA3, 2, 4-D and Ca foliar sprays on fruit chemical characteristics at the second harvest date EL2 Sugars (%) Treatment TSS Acidity TSS/ VC1 (mg/100 mL) (ppm) (%) (%) Acidity Reducing Non-reducing Total 2007/2008 Water 10.69c 0. 94b 11.37ab 56fg 304a 3.26d 4.39b 7.65d PUT 11.62cd 1.08ab 10.76bc 66c 262b 3.76bc 4.67ab 8.43c 2,4-D 10.93f 0.86b 12.71a 59e 248b 3.60cd 4.80ab 8.40c GA3 12.03b 1.18a 10.19c 68b 278a 3.74c 5.04ab 8.78bc Ca 11.82bc 1.16a 10.19c 65c 284a 3.78bc 5.14ab 8.92bc PUT +2,4-D 11.45d 0.96b 12.31ab 54g 268b 3.68cd 5.26a 8.94bc PUT + GA3 12.28a 1.12a 10.96bc 68b 253b 3.76bc 4.78ab 8.54c PUT + Ca 12.18a 0.96b 12.69a 73a 277ab 3.94bc 4.86ab 8.80bc 11.24e 0.92b 12.21ab 58ef 287a 4.66a 4.96ab 9.62a 2,4-D +GA3 2,4-D + Ca 12.06ab 0.96b 12.56a 59ef 289a 4.68a 5.25a 9.93a 12.28a 1.14a 10.77bc 64d 289a 4.46a 4.98ab 9.44a GA3 + Ca Water PUT 2,4-D GA3 Ca PUT +2,4-D PUT + GA3 PUT + Ca 2,4-D +GA3 2,4-D + Ca GA3 + Ca
1 2

11.14c 12.08ab 12.66a 11.86b 12.28a 12.14ab 11.96b 12.42ab 11.82b 12.16ab 12.20ab

1.02 0.93 0.98 0.89 0.84 0.90 0.94 0.86 0.80 1.00 0.89

10.92d 12. 99bc 12.92bc 13.33b 13.64 13.49b 12.72bc 14.44a 14.77a 12.16c 13.71ab

52f 59c 49a 67a 64b 50fg 60c 64b 55e 56d 65a

2008/2009 282a 260c 294a 257c 273b 248c 260c 265b 270b 261c 274b

3.67c 3.88c 3.89c 4.02b 4.45b 4.47b 4.36b 4.28b 4.66a 4.75a 4.66a

4.21c 4. 68b 4.86ab 4.78ab 4.88ab 5.00ab 4.94ab 4.87ab 5.08ab 5.17a 5.06ab

7.88f 8.56e 8.75de 8.80d 9.33bc 9.47b 9.30bc 9.15c 9.74a 9.92a 9.72a

Vitamin C, Electrolyte leakage. Values within a column with same letter are not signicantly different (P<0.05).

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange only. Electrolyte leakage was signicantly decreased in the rst season by spraying PUT alone, 2, 4-D alone, PUT + 2, 4-D and PUT + GA3. Whereas, in the second season it decreased in all treatments (except 2,4-D alone) as compared with the control. In both seasons, fruit reducing sugars content was signicantly increased by all sprays (except 2, 4-D alone in both seasons, PUT + 2,4-D in the rst season and PUT alone in the second season). In addition only foliar sprays of PUT + 2,4-D and 2,4-D + Ca resulted in higher non reducing sugars content than the control in the rst season, whereas, all foliar sprays signicantly increased non reducing sugars content in the second season. Total sugars content increased signicantly by all treatments in both seasons. Moreover, spraying 2, 4-D + GA3, 2,4-D + Ca and GA3 + Ca resulted the highest total sugars content when compared with all other treatments in both seasons. The data of both seasons indicated that all foliar sprays (except 2,4-D alone and 2,4-D + Ca in both season and 2,4-D + GA3 in the second season) decreased fruit carotenoids content as compared with the control. The improved fruit outer characteristics obtained for navel orange fruits in our study by the sprayed substances reected better fruit internal characters at both harvest dates. Juice TSS and sugars contents were increased. It is reported that at the late stages of citrus fruit development, soluble solids accumulate in the juice sacs (Coggins, 1981). Similar increases in TSS, VC and sugar contents were reported in Clementine mandarin (El-Otmani et al., 2004); Fortune mandarin (EL-Hilali et al., 2004) and Blood Red orange (Saleem et al., 2008). Ethylene induced fruit coloration and increased carotenoids content in Navel oranges (Rodrigo and Zacarias, 2007), so it might be concluded that putrescine and GA3 sprays inhibited ethylene production and then decreased carotenoids content and lowered fruit coloration. In conclusion, the improvement, we recorded in the external and internal fruit characteristics even when harvest date was delayed, indicates that GA3, PUT, Ca and 2, 4-D sprays might enable on-tree storage and late harvest by the benet of their combined effect on delaying development of the abscission layer, keeping the phloem and xylem connections in better condition, delaying fruit coloration, thus delaying fruit senescence. This might allow navel orange growers to have longer harvest season with only modest losses from fruit drop and without risks of fruit quality loss and thus extend export season in Egypt.

61

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62

Effect of putrescine, GA3, 2, 4-D and calcium on postharvest behaviour of navel orange
Sexton, R. and J.A. Roberts, 1982. Cell biology of abscission. Ann. Rev. Plant Physiol., 33: 133-162. Storey R., M.T. Treeby and D.J. Milne, 2005. Crease another calcium deciency-related fruit disorder. J. Hort. Sci. Biotechnology, 77: 565-571. Treeby, M.T. and R. Storey, 2002. Calcium spray treatments for ameliorating albedo breakdown in navel oranges. Australian Journal of Experimental Agriculture, 42(4): 495-502. Tumminelli, R., F. Conti, U. Maltese, C. Pedrotti and E. Bordonaro, 2005. Effects of 2, 4-D, 2, 4-DP, Triclopir and GA3 on pre-harvest fruit drop and senescence of Tarocco comune blood oranges in Sicilian orchards. Acta Hort., 682: 801-806. Valero, D., D. Martinez-Romero and M. Serrano, 2002, The role of polyamines in the improvement of the shelf life of fruit. Trends in Food Science Technology, 13: 228-234. Valero, D., D. Martinez-Romero, M. Serrano and F. Riquelme, 1998a. Polyamine response to external mechanical bruising in two mandarin cultivars. Hortscience, 33: 1220-1223. Valero, D., D. Martinez-Romero, M. Serrano and F. Riquelme, 1998b Inuence of postharvest treatment with putrescine and calcium on endogenous polayamines, rmness, and abscisic acid in lemon (Citrus lemon L. Burm cv. Verna). J. Agric. Food Chem., 46(6): 2102-2109. Valero, D., D. Martinez-Romero, M. Serrano and F. Riquelme, 1999. Polyamines roles on the post-harvest of fruits: A review. In: Recent Research Developments in Agricultural and Food Chemistry. S. Pandalai (Ed), Research Signpost, Trivandrum, India, pp. 39-55. Wimwright, H. and B. M. Burbage, 1989. Physiological disorders in mango (Mangifera indica L). Fruit J. Hort. Sci., 64: 125-135. Yang, S. F. and N. E., Hoffman, 1984. Ethylene biosynthesis and its regulation in higher plants. Annual Review Plant Physiol., 35:155189. Zarembinsiki, T. I. and A., Theologis, 1994. Ethylene biosynthesis and action: A case for conservation. Plant Mol. Biol., 26: 1579-1597. Zheng, Y. and Q., Zhang, 2004. Effects of polyamines and salicylic acid on postharvest storage of Ponkan mandarin. Acta Hort., 632: 317-320.
Received: January, 2010; Revised: June, 2011; Accepted: October, 2011

Martinez-Romero, D., M. Serrano, A. Carbonell, L. Burgos, F. Riquelme and D. Valero, 2002. Effects of postharvest putrescine treatment on extending shelf life and reducing mechanical damage in apricot. J. Food Sci., 67: 1706-1712. Messiaen J., P. Cambier and P. Van Cutsem, 1997. Polyamines and pectins. Plant Physiol., 113: 387-395. Moran, R. and D. Porath, 1980. Carotenoids determination in intact tissues. Plant Physiol., 65: 479. Paliyath, G. and M.J. Droillard, 1992. The mechanism of membrane deterioration and disassembly during senescence. Plant Physiol. Biochem., 30: 437-812. Pandey, S., S.A. Ranade, P.K. Nagar and N. Kumar, 2000. Role of polyamines and ethylene as modulators of plant senescence. Journal Bioscience, 25: 291-299. Perez-Vicente, A.D. Martinez-Romero, A. Carbonell, M. Serrano, F. Riquelme, F. Guillen and D. Valero, 2002. Role of polyamines in extending shelf life and the reduction of mechanical damage during plum (Prunus salicina Lind) storage. Postharvest Biology Technology, 25: 25-32. Pozo, L., W.J. Kender, J.K. Burns, U. Hartmond and A. Grant, 2000. Effects of gibberellic acid on ripening and rind pufng in Sunburst mandarin. Proc. Fla. State Hort. Soc., 113: 102-105. Rodrigo, M.J. and L. Zacarias, 2007. Effect of postharvest ethylene treatment on carotenoid accumulation and the expression of carotenoid biosynthetic genes in the avedo of orange (Citrus sinensis L. Osbeck) fruit. Postharvest Biology Technology, 43 (1): 14-22 Saftner, R.A. and B.G. Baldi, 1990. Polyamine levels and tomato fruit development: possible interaction with ethylene. Plant Physiology, 92: 547-550. Saleem, B.A., A.U. Malik, M.A. Pervez, A.S. Khan and M.N. Khan, 2008. Spring application of growth regulators affects fruit quality of Blood Red sweet orange. Pak. J. Bot., 40(3): 1013-1023. SAS Institute, SAS/STAT users guide, version 6.03, SAS Institute, Cary, NC, 1988 Sayed R. A., A.Z. Sabh and A.I. Ibrahim, 2004. Effect of calcium and gibberellic acid sprays on yield, quality and abscission of grape fruit. J. Agric. Sci. Mansoura Univ., 29(3): 1239-1255.

Journal

Journal of Applied Horticulture, 14(1): 63-66, 2012

Appl

In vitro free radical scavenging activity of aonla (Emblica ofcinalis) varieties at various stages of fruit development
S. Haripriya*, E. Vadivel, R. Venkatachalam and P. Gayathri1 Horticultural College and Research Institute, 1Department of Biochemistry, Centre for Plant Molecular Biology & Bioinformatics, Tamil Nadu Agricultural University 641 003, Tamil Nadu, India. *E-mail: hpshanmugam@gmail.com

Abstract
An investigation was undertaken to assess the free radical scavenging activity of aonla (Emblica ofcinalis) varieties viz., BSR-1, Chakaiya, Krishna and NA-7 at various stages of fruit development viz., initial stage, one-fourth maturity stage, half maturity stage, three-fourth maturity stage and full maturity stage using DPPH assay to identify the variety and stage of fruit development for maximum antioxidant activity. The experimental DPPH assay revealed that the free radical scavenging activity was signicantly different among the aonla varieties and also at various stages of fruit development in each variety. It was also found that the DPPH free radical scavenging activities of fresh aonla fruit extracts were found to be signicantly higher (P<0.05) than the radical scavenging activity of the standard ascorbic acid at varying concentrations. The pattern of total soluble sugars accumulation and free radical scavenging activity at various stages of fruit development in each aonla variety studied were discussed in detail. Key words: Aonla, free radical scavenging, DPPH, ascorbic acid, total soluble sugars.

Introduction
Oxidative stress has been identied to be the root cause of several chronic degenerative diseases, which occurs as a condition when the formed free radicals are not neutralized within our body system. Studies have proved that free radicals play an important role in pathogenesis of chronic degenerative diseases including cancer, diabetes, autoimmunity, inammatory, cardiovascular, neurodegenerative diseases and aging (Cantuti-Castelvetri et al., 2000; Surh et al., 2001; Vaya and Aviram 2001; Aruoma, 2003). Antioxidants are known to break the free radical chain reaction and scavenge the free radicals. A great interest has been recently focused on the natural foods, medicinal plants and phyto-constituents due to their well-known abilities to scavenge free radicals (Kukic et al., 2006; Galvez et al., 2005; Uma Nath and Deepak, 2009; Rezaeizadeh et al., 2011). Aonla ( Emblica officinalis) fruit of Euphorbiaceae family often referred to as Indian gooseberry, is one of the richest known sources of vitamin C. The fruits are reported to play an important role in scavenging free radicals (Tewari et al., 1982). High antioxidant activity of the aonla fruit was mainly due to the presence of ascorbic acid in higher percentage (Scartezzini et al., 2006; Ebrahimzadeh et al., 2011). Aonla fruits are a key constituent of many herbal preparations such as chyvanprash and triphala. The fruits have also been used in traditional medicinal systems, such as Chinese herbal medicine, Tibetan medicine and Indian medicine (Zhang et al., 2000) for centuries. The fruits of aonla are reported to contain hydrolysable tannins like emblicaninA and emblicanin-B, along with pedunculagin and punigluconin (Ghosal et al., 1996). Recent research and clinical studies indicate that it is these tannoid agents, especially emblicanin-A and emblicanin-B that are responsible for aonlas effectiveness as an antioxidant, anti-diabetic and anti-hyperlipidemic activities (Raghu et al., 2007).

Barring the discovery of ascorbic acid and the presence of large amount of tannins in aonla fuits, there does not seem to have any work done in line to assess its free radical scavenging activity in predominant Indian varieties and at various stages of its fruit development. There are many methods to evaluate the free radical scavenging activity of the tested compounds (Paulova et al., 2004). One of the widely used detection procedures, which facilitate analysis of various antioxidants, is based on 2, 2diphenyl-1- picryl hydrazyl radical (DPPH) bleaching (Bondet et al., 1997). Hence the present study was carried out to evaluate the in vitro DPPH free radical scavenging activity of the fresh fruit extract collected at various stages of fruit development in the leading varieties of aonla.

Materials and methods

The investigation was carried out at the Micro analytical laboratory of Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore. Source of aonla fruits: Aonla fruits of ve varieties (V), viz., BSR-1 (V1), Chakaiya (V2), Kanchan (V3), Krishana (V4) and NA-7 (V5) were freshly collected at ve different stages of fruit development (S) viz., initial stage (S1), one-fourth maturity stage (S2), half maturity stage (S3), three-fourth maturity stage (S4) and full maturity stage (S5) from 10 years old aonla trees grown in the orchard of Horticultural College and Research Institute, Coimbatore during July to November. Chemicals: DPPH (1,1-diphenyl-2-picrylhydrazyl) from M/s. Sigma-Aldrich Chemicals, Bangalore; Ascorbic acid standard from M/s. Merck, Mumbai and analytical grade methanol were used in this study. Preparation of aonla fruit extracts: 5 g of the fresh aonla fruit was weighed and macerated with 15 mL of methanol using pestle and mortar, which was then centrifuged at 3,000 rpm for

64

In vitro free radical scavenging activity of aonla varieties

Table 1. Comparison of free radical scavenging activity (%) of aonla varieties (V) at various stages of fruit development (S) Variety Free radical scavenging activity (%) S1 BSR-1 (V1) Chakaiya (V2) Kanchan (V3) Krishna (V4) NA-7 (V5) S- Mean 99.92 94.31 80.88 99.75 92.11 93.39 S2 99.97 94.31 84.09 99.79 96.90 95.01 S3 99.79 94.31 84.66 99.89 96.93 95.11 S4 94.74 93.31 79.48 99.63 96.29 92.69 S5 99.88 91.41 85.52 99.87 96.12 94.56 V-Mean 98.86 93.53 82.92 99.78 95.87

10 min. The supernatant containing the fruit extract was freshly prepared every time in this way for carrying out the experiment immediately after its collection from the orchard. Preparation of ascorbic acid: 1 mg of the ascorbic acid was dissolved in 1 mL of distilled water and different concentrations of ascorbic acid ranging from 10 g to 200 g were prepared accordingly. Experimental design: The experiment was carried out in Factorial Completely Randomized Design (FCRD) with two factors of Aonla variety (V) and various stages of fruit development (S). The numbers of days taken for fruit development in each stage of aonla with regard to its varieties are depicted in Fig.1. Determination of total soluble sugars (TSS): A hand held optical refractometer was used to measure the total soluble sugars of aonla varieties at various stages of fruit development. Evaluation of free radical scavenging activity using DPPH assay: The effect of aonla fruit extracts of major Indian varieties at various stages of fruit development on the DPPH free radical scavenging activity was assayed as per the method described by Hou et al. (2001). The scavenging of DPPH free radicals were monitored by recording the decrease in absorbance at 517 nm, which occurs due to the reaction with a radical species or reduction by the antioxidant. Methanol was used as the blank. 1 mL of distilled water along with 2 mL of DPPH served as the control. In this experiment, 50 L of aonla fruit extracts of ve varieties obtained at various stages of fruit development were taken in different test tubes. To this, 950 L of distilled water and 2 mL of DPPH were added, mixed well and incubated for 20 min at 37 C. Absorbance of the reaction mixture was recorded at 517 nm using spectrophotometer (SL159- Elico, UV-VIS). Ascorbic acid was used as the reference compound. The percentage scavenging (or) inhibition was calculated according to the formula, Percentage scavenging (or) Inhibition = (C- T / C) 100 Where, C is the absorbance of control and T is the absorbance of test. The experiment was replicated thrice and the data were

The SE(d) values of V(0.18), S (0.18) and VS(0.40); Similarly the CD (5 %) values of V(0.36), S(0.36) and VS (0.82). Table 2. DPPH radical scavenging activity (%) of ascorbic acid Concentration of ascorbic acid Free radical scavenging activity (g) (%) 10 20.40.4 20 39.80.2 40 86.70.5 60 92.40.1 80 95.70.2 100 96.20.4 120 96.50.3 140 96.60.1 160 96.40.2 200 96.40.2 The values are mean SD of the triplicates

analysed statistically as per procedure developed by Panse and Sukatme (1985).

Results and discussion

The 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical is widely used as a model system to investigate the free radical scavenging activities in several plant extracts. Nitrogen-centred, stable DPPH free radical produces violet colour in methanol solution and gets reduced to a yellow coloured product namely diphenyl picryl hydrazine, on addition of aonla fruit extracts. The experimental results recorded a signicant difference (P<0.05) in DPPH free radical scavenging activity among the aonla varieties investigated at various stages of fruit development (Table 1). A difference in DPPH free radical scavenging activity at varying concentration of the standard ascorbic acid (Table 2) was also observed. Among the varieties investigated, variety Krishna recorded signicant ( P< 0.05) DPPH free radical scavenging activity (99.78%) followed by the varieties BSR-1 (98.66%), NA-7 (95.87%), Chakaiya (93.53%) and Kanchan (82.92%). With regard to the stages of fruit development, stages S2 (95.01%) and S3 (95.11%) were on par in recording high free radical scavenging activity followed by stages S5 (94.56%), S1 (93.39%) and S4 (92.69%) respectively. Concerning the interaction between aonla varieties and the stages of fruit development, treatments V1S1 (99.92 %), V1S2 (99.97 %), V1S3 (99.79%), V1S5 (99.88%), V4S1 (99.75%), V4S2 (99.79%), V4S3 (99.89%) and V4S5 (99.87%) were found to be on par in recording high radical scavenging activity, while treatments V3S1 (80.88%) and V3S4 (79.48%) recorded the least radical scavenging activity. In the variety BSR-1, radical scavenging activity was found to

Fig. 1. Aonla varieties and number of days taken for fruit development in each stage. S indicates the various stages of fruit development in the investigated ve aonla varieties.

In vitro free radical scavenging activity of aonla varieties

65

be the highest during one-fourth stage of fruit maturity when compared to other stages of fruit development. This may be due to the presence of anthocyanins at early stages of fruit development, which was clearly evident from the dark red pigmentation present on the fruit surfaces and its disappearance at full maturity stage, wherein its activity decreases. This was in agreement with the finding that the concentration of pro-anthocyanins and total avonols were the highest in early fruit development stages in the craneberry varieties, Ben Lear and Stevens, during which their antioxidant activity was comparatively higher than the other stages (Vedenskaya and Vorsa, 2004). Variety Krishna exhibited a signicant consistency in its radical scavenging activity at all stages of fruit development compared to other investigated varieties. This may be due to a steady increase in TSS accumulation over the stages of fruit development, which in turn might have attributed to its consistancy in free radical scavenging activity. Radical scavenging activity gradually increased from initial stage to three-fourth maturity stage of fruit development, where it reached its peak activity and thereafter comparatively decreased at full maturity stage in the variety NA-7. A gradual increase in radical scavenging activity from initial to three-fourth stage of fruit development might have been contributed by a gradual increase in TSS accumulation in the fruit during these stages. In case of the variety Chakaiya, radical scavenging activity was on par from initial to half maturity stage of fruit development and then started to decrease gradually till it reached the full maturity stage. Amount of polyphenols and tannins accumulated during the early stages of fruit development might be higher, which was evidenced from its astringent taste. Astringency is an indicator for the presence of gallic acid and phyllembellin in higher levels, thereby its potency as free radical scavenger. The early fruit stage radical scavenging activities may be attributed to the astringency factor as a result of bitter principles accumulation. At later stages of fruit development, an increase in water content during TSS accumulation could have resulted in dilution of the accumulated phenolic substances, which might have reduced its radical scavenging activity. Among the varieties investigated, the least radical scavenging activity was observed in variety Kanchan, though its higher free radical scavenging activity was recorded at full maturity stage of fruit development. An average TSS content of 8.900 brix, accumulated over the stages of fruit development in the variety Kanchan was found to be relatively less when compared to the other varieties. In this regard, TSS was also found to play a very important role in ascorbic acid and polyphenol accumulations, which in turn might have affected its free radical scavenging activity. At full maturity stage, TSS accumulation in variety Kanchan was found to be comparatively higher effecting its higher radical scavenging activity. It is evident from Fig. 2 that TSS accumulation was highest in the variety BSR-1 followed by the varieties, Krishna, NA-7, Chakaiya and Kanchan. Peak TSS accumulation for each variety differed with regard to its stage of fruit development viz ., BSR-1 (one-fourth maturity), Chakaiya (initial to half maturity), Kanchan (full maturity), Krishna (half maturity) and NA-7 (full
BSR-1 Kanchan Krishna Chakaiya NA-7

Fig. 2. Comparison of total soluble sugars accumulation in aonla varieties at various stages of fruit development. Data shown are statistically analyzed at 5 % critical difference.

maturity). In all the aonla varieties investigated, a signicant decrease (P<0.05) in TSS accumulation was noticed in the three-fourth stage of maturity. In general, active sugar transport by means of substrate modification takes place during fruit development transitional stage. In case of aonla, this process might have taken place at three-fourth maturity stage of fruit development, wherein the substrate was chemically phosphorylated during the transport. While comparing the DPPH free radical scavenging activity of the standard ascorbic acid at varying concentrations with that of the aonla fresh fruit extract obtained from ve varieties at various stages of fruit development, it was clearly evident from Table 1 and Table 2 that the overall free radical scavenging activity of aonla was found to be higher than the standard (ascorbic acid) used in this experiment. There are some in vitro studies indicating that the antioxidant activities of aonla cannot be attributed to ascorbic acid alone and that the overall effect was also due to the presence of other polyphenols such as ellagic acid, gallic acid, tannins (Kabasakalis et al., 2000; Kim et al., 2005). It has also been found that some compounds in their natural formulations are more active than in their isolated form (Chrousos and Gold, 1992). This was found to be in accordance with the ndings of the experiment, while investigating DPPH free radical scavenging activity of predominant aonla varieties at various stages of fruit development. In aonla, a signicant difference in DPPH free radical scavenging activity among the varieties and at various stages of fruit development in each investigated variety was recorded. In

66

In vitro free radical scavenging activity of aonla varieties

Kukic, J., S. Petrovic and M. Niketic, 2006. Antioxidant activity of four endemic Stachys taxa. Biol. Pharm. Bul., 29: 725-729. Panse, V.G. and P.V. Sukhatme, 1985. Statistical Methods for Agricultural Workers. ICAR, New Delhi. Paulova, H., H. Bochorakova and E. Taborska, 2004. In vitro methods for estimation of the antioxidant activity of natural compounds. Chem. Listy., 98: 174-179. Raghu, V., Kalpana Patel and K.Srinivasan, 2007. Comparison of ascorbic acid content of Emblica ofcinalis fruits determined by different analytical methods. J. Food Comp. Anal., 20: 529-533. Rezaeizadeh, A., B.Z. Zuki, M. Abdollahi, Y.M. Goh, M.M. Noordin, M. Hamid and T.I. Azmi, 2011. Determination of antioxidant activity in methanolic and chloroformic extracts of Momordica charantia, Afr. J. Biotechnol., 10(24): 4932-4940. Scartezzini, P., F. Antognoni, M.A. Raggi, F. Poli and C. Sabbioni, 2006. Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation of Emblica ofcinalis Gaertn. J. Ethnopharm., 104: 113-118. Surh, Y.J., K.S. Chun, H.H. Cha, Y.S. Keum, K.K. Park and S.S. Lee, 2001. Molecular mechanisms underlying chemopreventive activities of anti-inammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-B activation. Mutat. Res., 480-1: 243-68. Tewari, S., M. Seshadri and T.B. Poduval, 1982. Migration inhibition of normal rat thymocytes as an in vitro method of detecting cell mediated immunity in rat and mouse. J. Immunol. Methods, 51: 231-239. Uma Nath, T. and C. Deepak, 2009. The plant extracts of Momordica charantia and Trigonella foenum graecum have antioxidant and anti-hyperglycemic properties for cardiac tissue during diabetes mellitus. Oxid. Med. Cell Longev., 2(5): 290-296. Vaya. J. and M. Aviram, 2001. Nutritional antioxidants mechanisms of action, analyses of activities and medical applications. Curr. Med. Chem., 1: 99-117. Vedenskaya, I.O. and N. Vorsa, 2004. Flavonoid composition over fruit development and maturation in American craneberry, Vaccinium macrocarpon. Plant Sci., 167(5): 1043-1054. Zhang, Y.J., T. Tanaka, Y. Iwamoto, C.R. Yang and I. Kouno, 2000. Phyllaemblic acid, a novel highly oxygenated norbisabolane from the roots of Phyllanthus emblica. Tetrahedron Lett., 41: 1781-1784.
Received: August, 2011; Revised: September, 2011; Accepted: December, 2011

addition, the free radical scavenging activity of fresh fruit aonla extract was found relatively higher than the standard ascorbic acid at varying concentrations. The ndings from this investigation could be commercially exploited in choosing the aonla varieties and harvesting fruits at the right stage of fruit development towards formulating natural antioxidant products from aonla fruits effecting high free radical scavenging activity. Future research may be directed towards the study of availability of antioxidants in the aonla varieties subjected to various processing methods.

References
Aruoma, O.I. 2003. Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mutat. Res., 9(20): 523-524. Bondet, V., W. Brand-Williams and C. Berset, 1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Food Sci. Technol., 30: 609-615. Cantuti-Castelvetri, I., B. Shukitt-Hale and J.A. Joseph, 2000. Neurobehavioral aspects of antioxidants in aging. Int. J. Dev. Neurosci., 18: 367-381. Chrousos, G.P. and P.W. Gold, 1992. The concepts of stress and stress-system disorders overview of physical and behavioural homeostasis. J. Am. Med. Assoc., 267: 1244-1252. Ebrahimzadeh, M.A., S.M. Nabavi, S.F. Nabavi and A.A. Dehpour, 2011. Antioxidant activity of hydroalcholic extract of Ferula gummosa Boiss roots. Eur. Rev. Med. Pharmacol. Sci., 15(6): 658-664. Galvez, M., C. Martin-Cordero, P.J. Houghton and M J. Ayuso, 2005. Antioxidant activity of methanol extracts obtained from Plantago species. J. Agric. Food Chem., 53: 1927-1933. Ghosal, S., V.K. Tripathi and S. Chauhan, 1996. Active constituent of Emblica ofcinalis: The chemistry and antioxidant effects of two new hydrolysable tannins, emblicanin A and B. Indian J. Chem., 35B: 941-948. Hou, W.C., Y.C. Chen, H.J. Chen, Y.H. Lin, L.L. Yang and M.H. Lee, 2001. Antioxidant activities of trypsin inhibitor, a 33 kDa root storage protein of sweet potato (Ipomoea batatus (L) Lam cv.Tainong 57). J. Agr. Food Chem., 49: 2978-2981. Kabasakalis, V., D. Siopidou and E. Moshatou, 2000. Ascorbic acid content of commercial fruit juices and its rate of loss upon storage. Food Chem., 70: 325-328. Kim, H.J., T. Yokojawa, H.Y. Kim, C. Tohda, T.P. Rao and L.R. Juneja, 2005. Inuence of aonla (Emblica ofcinalis) on hypercholesterolemia and lipid peroxidation in chloresterol fed rats. J. Nutr. Sci. Vitaminol., 51: 413-418.

Journal

Journal of Applied Horticulture, 14(1): 67-70, 2012

Appl

Response of some Egyptian sweet melon (Cucumis melo var. Aegyptiacus L.) cultivars to water stress conditions
E.A. Ibrahim Vegetable Research Department, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt. E-mail: e_ebraheem@yahoo.com

Abstract
Drought is a wide-spread problem, seriously inuencing sweet melon (Cucumis melo var. Aegyptiacus L.) production and quality. Therefore, identication or development of tolerant genotypes is of immense importance for sweet melon production in drought prone areas. Two eld experiments were conducted in clay loam soil at Baramoon Experimental Farm, Dakahlia Governorate, Egypt during the two summer seasons of 2008 and 2009, to evaluate ve sweet melon cultivars (Shahd El-Dokki, Ananas El-Dokki, Ismaelawi, Kahera-6 Improved, Albasosi) under regular irrigation and stress conditions (drought conditions were imposed after rst irrigation and created by reducing the frequency of irrigation by one half to that of irrigated crop, i.e., missing alternate irrigation) using a split plot design with three replicates. Drought susceptibility index, relative yield reduction and relative yield values were used to describe yield stability and yield potential. Results indicated that exposure of sweet melon cultivars to water stress lead to signicant decrease in fruit weight, fruit length, fruit width, fruit esh thickness and total yield per plant. Whereas, water decit caused signicant increase in total soluble solids. The tested cultivars markedly varied among them in all estimated characters. The interaction between irrigation levels and cultivars had signicant effects on all traits under study in both seasons. Cultivars with the highest yield and yield components under non-stress conditions had the highest yield and yield components under stress conditions. On the basis of the drought resistance indices, Kahera-6 Improved was relatively stress susceptible, whereas Albasosi was more tolerant and stable cultivar therefore detailed studies are warrented for validating its drought tolerance characterstic. Key words: Cucumis melo, sweet melon, cultivars, water stress, drought resistance.

Introduction
Sweet melon (Cucumis melo var. Aegyptiacus L.) is considered as one of the most important vegetable crops grown in Egypt. Fruits are consumed in the summer season and are popular because the pulp of the fruit is very refreshing, highly nutritional and sweet with a pleasant aroma (Melo et al., 2000). The shortage of water availability has become a worldwide problem; therefore, there has been an intense interest in studying plant water stress interactions in arid and semi-arid environments. In Egypt, under limited water supply conditions, the farmers tend to increase the irrigation interval, which creates water stress. Water stress is one of the most important factors affecting every aspect of plant growth. Many irrigation experiments have shown that melon is sensitive to water stress (Faberio et al., 2002; Sensoy et al., 2007). Fruit yield and its components were highly inuenced by the total volume of irrigation water at different crop stages in a semi-arid climate (Faberio et al., 2002). Water decit produces smaller fruits (Fabeiro et al., 2002; Long et al., 2006) and lower yields (Cabello et al., 2009; Dogan et al., 2008; Kirnak et al., 2005; Sensoy et al., 2007; Zeng et al., 2009). Although considerable variation for drought resistance has been identied among muskmelon cultivars (El-Kassas and ElSebsey, 1998), very little work has been done to study the effects of water stress on sweet melon cultivars in Egypt. In future years breeding programs must consider and select from newly released varieties with improved water use efciency. In this regard, screening for

more drought tolerant sweet melon varieties, which are able to produce an acceptable yield under water stress, is an important component in melon breeding research programs. Thus, it is important to identify sweet melon genotypes with high yield potential and stability under drought stress. This study was an attempt to measure some effects of moisture stress levels on ve sweet melon cultivars and to screen them for yield potential and stability under water stress conditions to identify cultivars that can adapt to water decit conditions.

Materials and methods

Two eld experiments were performed during the two summer seasons of 2008 and 2009 at Baramoon Experimental Farm, Dakahlia Governorate, Egypt, where the soil is clay-loam. Five sweet melon cultivars (Shahd El-Dokki, Ananas El-Dokki, Ismaelawi, Kahera-6 Improved and Albasosi) were used for this study. A split plot design with three replicates was used for experimental layout. The main plots were assigned to two irrigation levels (regular irrigation and stress conditions). Drought conditions were imposed after rst irrigation and created by reducing the frequency of irrigation by one half to that of irrigated crop, i.e., missing alternate irrigation. Sub plots were used for ve sweet melon cultivars. Each experimental unit area was consisted of four ridges (5 m in length and 1.5 m in width) and plants were spaced 50 cm apart.

68

Response of some Egyptians sweet melon cultivars to water stress conditions

Table 1. Effect of irrigation levels on sweet melon yield and its components during summer 2008 and 2009 seasons Irrigation Fruit levels weight (g) Fruit length (cm) Fruit Flesh width fruit (cm) thickness (cm) 2008 season 23.08 15.02 3.88 13.30 3.45 0.39 22.02 20.15 1.38 0.64 0.19 2009 season 14.13 12.99 0.15 3.67 3.20 0.10 TSS (%) Total yield / plant (g)

Seeds were sown on 7th and 6th April in both study seasons, respectively. Except irrigation, recomended cultural practices for sweet melon were followed. At the harvesting time, a random sample of 12 plants was taken from each experimental unit and data were recorded for fruit weight (g), fruit length (cm), fruit width (cm), esh fruit thickness (cm), total yield per plant (g) and total soluble solids (TSS). Total soluble solids were determined using a hand refractometer. The data were statistically analyzed according to Snedecor and Cochran (1982). Comparisons among means of treatments were tested using LSD values at 5% level. Evaluation of drought resistance: Based on average of two seasons, the results were used to evaluate the effect of drought stress. Drought resistance indices were dened by following formula:
Stress susceptibility index = (1-Ys/Yw)/D (Fisher and Maurer, 1978) Relative yield reduction = 1-Ys/Yw (Hiller and Clark, 1971)

Normal Stress LSD 5% Normal Stress LSD 5%

2429 1896 61 2304 1809 172

9.93 11.71 0.26 9.50 11.44 1.23

5779 4170 204 5642 4091 47

20.68

Table 2. Effect of sweet melon cultivars on yield and its components during summer 2008 and 2009 seasons Flesh Fruit Fruit Fruit fruit weight length width (cm) (cm) thickness (g) (cm) 2008 season Shahd El-Dokki 1354 19.21 11.66 3.30 Ananas El-Dokki 1540 17.11 13.32 3.37 Ismaelawi 2292 27.50 12.91 3.45 Kahera-6 Improved 1675 17.06 15.95 3.16 Albasosi 3952 28.53 16.96 5.05 LSD 5% 185 1.45 1.17 0.35 2009 season Shahd El-Dokki 1279 18.24 11.31 3.16 Cultivars Ananas El-Dokki Ismaelawi Albasosi LSD 5% 1435 2167 3831 155 16.46 26.53 16.55 27.65 1.07 12.77 12.40 15.10 16.20 1.00 3.26 3.32 3.05 4.88 0.19 TSS (%) Total yield / plant (g) 3280 3867 5135 4292 8298 223 3219 3833 5025 4203 8054 139

Where Ys is the mean of yield under drought, Yw is the mean of the same character under well-watered conditions, and D is the environmental stress intensity = 1-(mean yield of all varieties under drought/mean yield of all varieties under well-watered conditions). The relative yield under drought was calculated as the yield of a specic genotype under drought divided by that of the highest yielding genotype in the population.

Results and discussion

Effect of irrigation levels: Data illustrated in Table 1 reveals that the drought stress signicantly decreased the fruit weight, fruit length, fruit width, esh fruit thickness and total yield per plant in both the seasons. Under water decit, low crop yield obtained may be due to infrequent application of water resulting in a lack of moisture in active crop root zone, inadequate moisture conservation, and poor nutrient utilization (Frank and Viets, 1967). Therefore, it was concluded that if irrigation water is available, the sweet melon plants must be irrigated regularly to obtain higher yield. Whereas, water decit caused signicant increase in total soluble solids in both seasons. This result may be attributed to the decrement in the water content of the plant which caused a remarkable increase in the cell sap concentration. TSS is an important parameter for drought. A similar effect of drought on TSS was also observed by Fabeiro et al. (2002), Long et al., (2006), Sensoy et al. (2007) and Zeng et al. (2009). Effect of sweet melon cultivars: Table 2 reveals that sweet melon cultivars exhibited signicant differences for all characters in both the seasons. Albasosi cultivar gave the highest fruit weight, fruit length, fruit width, esh fruit thickness and total yield per plant, and had the lowest values of TSS than other cultivars in both seasons. While, Shahd El-Dokki cultivar gave the lowest values of fruit weight, fruit width, and total yield per plant in both seasons. The lowest values of fruit length and esh fruit thickness were recorded in cv. Kahera-6 Improved, however, fruits of Kahera-6 Improved had the highest values of TSS. These ndings were similar in both experimental seasons. Some investigators concluded that the genotypic variation between cultivars of Egyptians sweet melon might result in variation in yield and yield components (ElDweneny 1978; El- Shimi and Ghoneim, 2006).

10.48 11.48 9.95 12.95 9.23 0.82 10.10 11.32 9.58 12.40 8.94 0.49

Kahera-6 Improved 1571

Effect of the interaction between irrigation levels and cultivars: The interaction between irrigation levels and cultivars had signicant effects on all studied traits in both the seasons (Table 3). The results clearly show that for all sweet melon cultivars, the effect of drought stress resulted in reduction in all studied traits except TSS. Cv. Albasosi, watered regularly, gave the highest values for fruit weight, fruit length, fruit width, esh fruit thickness and total yield per plant, but it gave lowest value for TSS in comparison with other treatments in both seasons (Table 3). While stressed plants of cv. Shahd El-Dokki exhibited reduction in fruit weight, fruit width, and total yield per plant in both seasons. Highest values of TSS was recorded in stressed plants of cv. Kahera-6 Improved. These results were in agreement with El-Kassas and ElSebsey (1998) on muskmelon. Evaluation of drought resistance: Water stress induced yield decline is useful indicator for assessing drought resistance. Results in Table 4 implied that the highest yield reduction occurred for Kahera-6 Improved (29%). The lowest yield reduction was for Ananas El-Dokki (26 %). The stress susceptibility index (SSI) appeared to be a suitable selection index to distinguish resistant cultivars. Genotypes with

Response of some Egyptians sweet melon cultivars to water stress conditions

Table 3. Effect of the interaction between irrigation levels and cultivars on sweet melon yield and its components seasons Treatments Fruit weight Fruit length Fruit width Flesh fruit (g) (cm) (cm) thickness (cm) Irrigation Cultivars 2008 season Shahd El-Dokki 1519 20.39 12.50 3.48 Ananas El-Dokki 1712 18.01 14.13 3.56 Normal Ismaelawi 2587 28.87 13.66 3.68 Kahera-6 Improved 1875 18.04 16.87 3.31 Albasosi 4453 30.11 17.93 5.37 Shahd El-Dokki 1189 18.03 10.82 3.12 Ananas El-Dokki 1368 16.20 12.51 3.19 Stress Ismaelawi 1997 26.13 12.16 3.23 Kahera-6 Improved 1474 16.08 15.03 3.00 Albasosi 3450 26.95 15.98 4.72 LSD 5% 68 2.06 1.66 0.50 2009 season Shahd El-Dokki 1404 19.11 11.78 3.25 Ananas El-Dokki 1572 16.95 13.21 3.36 Normal Ismaelawi 2451 27.53 12.86 3.47 Kahera-6 Improved 1756 17.45 15.87 3.11 Albasosi 4337 29.07 16.93 5.14 Shahd El-Dokki 1153 17.36 10.84 3.06 Ananas El-Dokki 1298 15.97 12.32 3.15 Stress Ismaelawi 1883 25.54 11.95 3.17 Kahera-6 Improved 1386 15.66 14.34 2.98 Albasosi 3324 26.24 15.47 4.62 LSD 5% 219 1.51 1.41 0.27 Table 4. Average yields of ve sweet melon cultivars under normal (Yw) and stress (Ys) conditions, stress susceptibility index, Relative yield reduction and relative yield under water stress (RYS) Cultivars Total yield / Plant Relative RYS Stress (g) yield susceptibility index Yw Ys reduction 3757 2742 27 0.98 0.40 4426 5922 4976 9472 5711 3273 4237 3519 6931 4140 26 28 29 27 0.95 1.03 1.06 0.99 0.47 0.61 0.51 1.00 0.60

69
during summer 2008 and 2009 TSS (%) 9.53 10.71 9.15 12.01 8.25 11.43 12.24 10.75 13.89 10.22 1.16 9.22 10.19 8.68 11.25 8.14 10.98 12.46 10.48 13.56 9.74 0.69 Total yield / plant (g) 3813 4485 5995 5044 9558 2747 3248 4274 3540 7038 316 3701 4367 5849 4907 9385 2737 3298 4200 3498 6723 197

It is concluded that Albasosi is a more tolerant variety among the studied varieties and could be further tested for other drought conferring characteristics. Kahera-6 Improved with the highest SSI and relative yield reduction with low relative yield under water stress condition was identied as sensitive cultivar.

Shahd El-Dokki Ananas El-Dokki Ismaelawi Kahera-6 Improved Albasosi Mean

References
Cabello , M.J., M.T. Castellanos, F. Romojaro, C. Martnez-Madrid and F. Ribas, 2009. Yield and quality of melon grown under different irrigation and nitrogen rates. Agric. Water Manage., 96: 866-874. Dogan, E., H. Kirnak, K. Berekatoglu, L. Bilgel and A. Surucu, 2008. Water stress imposed on muskmelon (Cucumis melo L.) with subsurface and surface drip irrigation systems under semiarid climatic conditions. Irrig. Sci., 26(2): 131-138. El-Deweny, H.H.A. 1978. Evaluation of Some Varieties of Sweet Melon and Muskmelon. Ph. D. Thesis, Fac. Agric. Ain Shams Univ., Egypt, 130 pp. El-Kassas, A.I. and A.A. ElSebsey, 1998. Effect of irrigation regimes and foliar potassium fertilizer on yield of muskmelon in north Sinai. J. Agric. Sci. Mansoura Univ., 23(6): 2867-2877. El-Shimi, I.Z.A. and M.I. Ghoneim, 2006. Evaluation of morphological and pathological performance for some local melon landraces. The Fourth Conference on Scientic Research Outlook &. Technology Development in the Arab World 11-14 December. TuA6 623, Umayyad Palace, Damascus, Syria. Fabeiro, C., F. Martn and J.A. de Juan, 2002. Production of muskmelon (Cucumis melo L.) under controlled decit irrigation in a semi-arid climate. Agric. Water Manage., 54: 93-105. Fisher, R.A. and R. Maurer, 1978. Drought resistance in spring wheat cultivars: I. Grain yield responses. Aust. J. Agric Res., 29: 897-912.

low SSI values (less than 1) can be considered to be drought resistant (Bruckner and Frohberg, 1987), because they exhibited smaller yield reductions under water stress compared with wellwatered conditions than the mean of all genotypes. The cultivars Kahera-6 Improved and Ismaelawi were relatively drought susceptible (SSI > 1), while the cultivars Shahd El-Dokki, Ananas El-Dokki and Albasosi were relatively drought resistant (SSI values < 1). However, the low SSI values may not necessarily give a good indication of drought resistance of a genotype. Low SSI values of a variety could be due to lack of yield production under well-watered conditions rather than an indication of its ability to tolerate water stress. Therefore, a stress tolerant genotype as dened by SSI, need necessarily not to have a high yield potential. Cultivar Albasosi was relatively high yielding under water stress (RY > mean RY), while Shahd El-Dokki, Ananas and El-Dokki were relatively low yielding (RY < mean RY).

70

Response of some Egyptians sweet melon cultivars to water stress conditions

Melo, M.L.S., N. Narain and P.S. Bora, 2000. Characterisation of some nutritional constituents of melon (Cucumis melo hybrid AF-522) seeds. Food Chem., 68: 411-414. Sensoy, S., A. Ertek, I. Gedik and C. Kucukyumuk, 2007. Irrigation frequency and amount affect yield and quality of eld grown melon (Cucumis melo L.). Agric. Water Manage., 88: 269-274. Snedecor, G.W. and W.G. Cochran, 1982. Statistical Methods. Seventh Edition, 2nd Printing. Iowa State Univ. Press, Ame., USA, 507 PP. Zeng, C., Z. Bie and B. Yuan, 2009. Determination of optimum irrigation water amount for drip-irrigated muskmelon (Cucumis melo L.) in plastic greenhouse. Agric. water manage., 96: 595-602.
Received: December, 2010; Revised: August, 2011; Accepted: December, 2011

Frank, G. and J.R. Viets, 1967. Nutrient availability in relation to soil water. In: Irrigation of Agricultural Lands, R.M. Hagan, H.R. Haise and T.W. Edminster (eds.). American Society of Agronomy, Publisher Madison, Wisconsin, USA. Hiller, E.A. and R.N. Clark, 1971. Stress day index to characterized effects of water stress on crop yields. Trans. ASAE, 14: 757. Kirnak, H., D. Higgs, C. Kaya and I. Tas, 2005. Effects of irrigation and nitrogen rates on growth, yield, and quality of muskmelon in semiarid regions. J. Plant Nutr., 28: 621-638. Long, R.L., K.B. Walsh and D.J. Midmore, 2006. Irrigation scheduling to increase muskmelon fruit biomass and soluble solids concentration. Hortscience, 41(2): 367-369.

Journal

Journal of Applied Horticulture, 14(1): 71-73, 2012

Appl

Micropropagation of strawberry cultivar Sweet Charlie through axillary shoot proliferation

R. Rekha, Pallavi Mandave and Neelambika Meti* Department of Plant Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University, Pune- 4110046, Maharashtra, India. *E-mail: neelambika.meti@gmail.com

Abstract
A protocol for micropropagation of strawberry cv. Sweet Charlie was standardized through axillary shoot proliferation from runner tips. Medium supplemented with TDZ (1 mg/L) alone was favourable for the induction of multiple shoots and daughter runners from runner tips. Such shoots were successfully multiplied for four times on MS incorporated with 0.5mg/L each of BAP, IBA and 1.0 mg/L of GA3. Rooting of subcultured shoots was achieved on MS medium containing 0.5 mg/L of kinetin alone and along with 0.5 mg/L of IBA. Ex agar plants were harvested regularly after three weeks of growth period for their acclimatization in both cocopeat and soil. The survival rate of tissue cultured plants was 85%. Key words: Axillary shoot proliferation, strawberry, runner tips, TDZ, daughter runners

Introduction
The cultivated strawberry (Fragaria x ananassa Duch.) is an octaploid species (2n=8x=56) belonging to genus Fragaria of family Rosaceae with at least 15 species (Hancock, 1990). Their fruits are rich in bioactive phytochemicals, especially phenolic compounds with high antioxidant capacity, and are known to be benecial to human health when they are consumed as part of daily diet (Hannum, 2004). These nutritional qualities have ensured economic importance of this crop throughout the world and currently this crop is of primary interest for both research and fruit production. The cv. Sweet Charlie is a hybrid between FL80-456 and Pajaro and tested as FL 85-4925 and is popular for its delicious, sweet and nutritious fruits. In vitro techniques are important tools for modern plant improvement programs to introduce new traits into selected plants, to multiply elite selections and to develop suitable cultivars in a minimum time. Moreover, the ability to regenerate plants is crucial to the successful application of in vitro methods (Taji et al., 2002). Strawberries are vegetatively propagated by runners arising from axillary buds on the plant crown in a limited number. The tissue culture plants seemed to produce more runners per mother plant in a short time (Mohan et al., 2005). Plants produced by axillary branching normally retain the genetic composition of the mother plant and this method has proven to be the most applied and reliable method for the production of true-to-types. Successful plant regeneration has been obtained in strawberry from single meristems (Boxus, 1999), node culture (Bhatt and Dhar, 2000), runner tips (Litwinczuk, 2004) and shoot tips (Ko Chien Ying et al., 2009). Thidiazuron (TDZ), a substituted phenylurea (N phenyl-N-1,2,3-thidiazol-5-yl urea) with its cytokinin- and auxin-like effects, is now among the most active cytokinin like substances (Huetteman and Preece, 1993) used to induce axillary shoot proliferation, shoot organogenesis either alone or in combination with auxins (Passey et al., 2003) in strawberries. An efcient method of micropropagation technique however deemed

essential for supply of reliable plant material of this cultivar to meet the demands of strawberry growers from Mahabaleshwar hill region in Maharashtra. Though, Sweet Charlie is largely cultivated and well adapted variety in India, it is less studied variety. The objective of this study was to investigate the feasible protocol for micropropagation of strawberry cv. Sweet Charlie.

Material and methods

Strawberry plants were collected from Strawberry Growers Association, Mahabaleshwar and maintained in institutes shade net for further experimentation. Runner tips from the mother plants of strawberry cv. Sweet Charlie were collected from plants maintained in shadenet house of the institute and were treated with Bavistin (2%) for 2 min. followed by surface sterlization with 0.1% mercuric chloride for 1 min. After repeated wash in sterile distilled water and 1 min treatment with 70% absolute alcohol, explants were reared on Murashige and Skoogs medium (Murashige and Skoog, 1962, MS) with sucrose (3%) and agar (0.8%) alone and along with TDZ 0.5, 1.0 and 2.0 mg/L individually and in a different combinations with each of 0.2 and 0.5 mg/L BAP and KN, 0.5 mg/L of IBA, 1.0 mg/L of GA3. The pH of the medium was adjusted to 6.0. All the cultures were maintained at 26 oC (under 1000 lux) RH 50-60% at 12 hours photoperiod. For each treatment, 12 replicates were used in triplicate. The cultures were maintained for a period of three weeks and at the end of each experiment, cultures/calli were subcultured on a fresh medium for their growth and development. The shoots, roots and somatic embryos were observed using a stereoscopic microscope. The cultures were photographed wherever necessary. The regenerated plantlets via axillary shoot proliferation were successfully shifted to primary hardening in portrays lled with sterilized cocopeat after rinsing in 0.2% Bavistin. The protrays were covered with perforated polythene bags to maintain humidity for two weeks. During third week, the plants were shifted to polybags lled with soil:sand:cocopeat (1:1:1) and were gradually exposed to shadenet condition.

72

Micropropagation of strawberry cultivar Sweet Charlie through axillary shoot proliferation

Results
The runner tips were inoculated on MS medium containing two concentration of TDZ (0.5 and 1.0 mg L-1). The induction of multiple shoots occured within 6 weeks from the runner tips along with the formation of daughter runners (Fig 1a) and the responses are shown in Table 1. The maximum number of stunted shoots, per explants were observed on MS medium supplemented with 1 mg L-1 TDZ. The separated multiple shoots from induction medium were subcultured on different media containing lower concentration of BAP and KN individually each at 0.5 mg/L and in combination with GA3 (1 mg/L) and/ or IBA (0.5 mg/L) and the responses are shown in Table 2. The proliferation of multiple shoots was noticed on the medium supplemented with BAP alone and along with GA 3 and /or IBA or KN (Fig. 1b).Whereas, addition of BAP or KN individually and along with GA3 supported elongation of shoots. On the contrary, incorporation of IBA (0.5 mg/L) supported the growth of both multiple shoots and their elongation (Fig. 1c). Ex agar plants (Fig. 1d) were successfully hardened by gradually lowering the humidity over 3 weeks in cocopeat as protray plants (Fig. 1e) and in soil as secondary hardened plants (Fig. 1f) after regular three weeks interval period.
Table 1. Response of runner-tip cultures on Fig.1. Micropropagation of strawberry cv. Sweet Charlie from runner tips. a) Runner tip with multiple MS supplemented with TDZ in strawberry shoots from clump and in vitro runner. b) Separated shoots on multiplication medium. c) Regenerated cv. Sweet Charlie shoots on rooting medium ready for harvesting TDZ Cultures Number of variety is not available. We used TDZ for the rst time to get plants from axillary shoots. mg L-1 responded (%) shoots/ explant Explants taken from eld-grown plants are difcult to sterilize to establish in vitro cultures 0.0 50 1.0 due to high degree of contamination. It is usually recommended to take explants from 0.5 50 11.0 plants grown under controlled conditions such as growth room or greenhouse to obtain 1.0 95 20.2

better results. In the present study, individual TDZ effect was responsible for multiple shoot induction along with daughter runners from runner tips collected from green house Discussion maintained mother plants. In vitro runner formation was earlier reported in cvs. Gorella In India, strawberry are cultivated largely and Hakras Romata on Boxus medium with or without GA3 (Zatyk et al., 1989). The in many places. Though cv. Sweet Charlie present study also demonstrates formation of daughter runners on MS medium with TDZ. is cultivated but literature related to this In cv. Camarosa, report on signicant effect of TDZ alone and in combination with IAA
Table 2. Response of subcultured shoots for plant regeneration or multiple shoots on MS medium supplemented with different types of PGR PGR level (mg/L) Shoots/ Response type Cultures with multiple shoots culture (%) BAP KN GA3 IBA 0.5 1.0 Root induction and regeneration of plantlets 11.3 0.5 1.0 Root induction and regeneration of plantlets 16.6 0.5 1.0 1.0 Elongation of shoots 14.3 0.5 1.0 1.0 Elongation of shoots 19.7 0.2 0.2 1.0 2.0 Shoot multiplication and their elongation 55.3 0.5 1.0 0.5 4.0 Shoot multiplication and their elongation 90.7

Micropropagation of strawberry cultivar Sweet Charlie through axillary shoot proliferation for shoot proliferation, as well as at the rooting stage, addition of 1.0 mg/L of TDZ along with charcoal and IAA has been reported (Adak et al., 2009). On the contrary to earlier reports on three strawberry clones other than Sweet Charlie, the highest response of shoot multiplication from nodal segments at the rate of three was obtained in MS containing lBA and KN and the maximum frequency of rooting was produced on medium containing 1.0 mg /L IBA (Sakila et al., 2007). The present study revealed the potential of successful year round multiplication of strawberry cv. Sweet Charlie from nodal segments to meet the demand for quality planting material in strawberry growing areas.

73

Acknowledgements
Authors thank Former Principal, Late Dr. R.M. Kothari for his encouragement, Dr. E.A. Singh and Dr. Bipinraj, for assistance in collection of plant material and Professors G.D. Sharma and P.K. Ranjekar for critically going through the MS. The nancial support from Bharati Vidyapeeth University, Pune, Maharashtra, India is gratefully acknowledged.

References
Adak, N., L. Kaynak, M. Pekmezci and H. Gubbuk, 2009. The effect of various hormone types on in vitro propagation of strawberry. Acta Hort., 829: 305-308. Bhatt, I.D. and U. Dhar, 2000. Micropropagation of Indian wild strawberry. Plant Cell, Tissu. Org. Cult., 60: 83-88. Boxus, P. 1999. Micropropagation of strawberry via axillary shoot proliferation. Methods Molecular Biology, 111: 103-114. Hancock, J.F. 1990. Ecological genetics of natural strawberry species. HortScience, 25(8): 869-871.

Hannum, S.M. 2004. Potential impact of strawberries on human health: a review of the science. Crit. Rev. Food Sci. Nutr., 44: 1-17. Huetteman, B. and E.J. Preece, 1993. Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell. Tiss. Org. Cult., 33: 105-119. Ko Chien-Ying, A.M. Al-Abdulkarim, S.M. Al-Jowid and A.Al-Baiz, 2009. An effective disinfection protocol for plant regeneration from shoot tip cultures of strawberry. Afri. J. of Biotech., 8(11): 2611-2615. Litwinczuk, W. 2004. Field performance of Senga Sengana strawberry plants (Fragaria ananassa) obtained by runners and in vitro through axillary and adventitious shoots. Electronic J. Polish Agri. Uni., 7(1): 1505-0297. Mohan, R., E.A. Chui, L.A. Biasi and C.R. Soccol, 2005. Alternative in vitro propagation: use of sugarcane bagasse as a low cost support material during rooting stage of strawberry cv. Dover. Braz. Archives Bio. Techn., 48: 37-42. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture., Physiol. Plant., 15: 473-497. Passey, B.J., K.J. Barrett and D.J. James, 2003. Adventitious shoot regeneration from seven commercial strawberry cultivars (Fragaria ananasse Duch.) using a range of explant types. Plant Cell Reports, 21: 397-401. Sakila, S., M.B. Ahme, U.K. Roy, M.K. Biswas, R. Karim, M.K. Razvy and M. Hossain, 2007. Micropropagation of strawberry (Fragaria x ananasse Duch.): A newly indroduced crop in Bangladesh. Ame.Eurasian Jour. Sci. Res., 2(2): 151-154. Sood, N., R. Srivastava, O.S. Singh and S.S. Gosal, 2000. Enhancing micropropagation efciency of strawberry using bandage in liquid media. Jour. Appl. Hort., 2(2): 92-93. Taji, A., P.P. Kumar and P. Lakshmanan, 2002. In vitro Plant Breeding, Food Products Press, New York, 167. Zatyk, J.M., G. Kiss, Zs. Radics and I. Simon, 1989. Initiation of strawberry runner formation in vitro. Acta Hort., 265: 349-352.
Received: October, 2010; Revised: November, 2011; Accepted: December, 2011

Journal

Journal of Applied Horticulture, 14(1): 74-76, 2012

Appl

Potential use of shea nut (Vitteleria paradoxom) butter as skin coat for ripening and improved storage of banana
E.K. Tsado School of Agriculture and Agricultural Technology, Federal University of Technology, P.M.B., 65. Minna Niger State, E- mail: ektsado@yahoo.co.uk

Abstract
The study was designed to assess the effect of locally produced butter from nuts of shea butter trees (Vitteleria paradoxom) on the ripening and storage of banana. A simple complete randomized experimental design was used to test the effect of coating matured banana ngers with shea butter oil before storage under three temperature conditions viz., 35, 25 and 10 oC. Each treatment was replicated three times. Results showed a signicant effect of different storage temperatures. Days to ripening between coated and uncoated bananas, and the interaction with storage temperatures were not statistically different. A taste panels results of assessing the effect of coating treatment on the textual quality of ripe bananas did not show any signicant difference neither was there an effect on the appeal of ripened bananas. The result showed that banana ngers stored in the refrigerator at 10oC lasted beyond 53 days of storage irrespective of the treatment. At 25 oC, the coated ngers took 15.7 days to ripen while the uncoated lasted 8 days. Coated banana ngers stored at 35 oC took 11.3 days to ripen but the uncoated ripened after 6 days. The use of shea butter for shelf life prolongation is discussed while the test is continuing. Key words: Shea butter tree (Vitellaria paradoxa), banana, ripening, storage, wax coating

Introduction
The shea butter tree (Vitellaria paradoxa C. F. Gaertn.) is a very important tree in West Africa especially in central Nigeria because of its high prospective contribution to the reduction of poverty, hunger and disease among rural people. It serves as efcient fuel wood. Its fruit pulp has excellent nutritional content (Ugese et al., 2008) and is widely consumed among indigenous peoples of central Nigeria (Maranz et al., 2004). In Nigeria, its oil is used as a cooking fat and in chocolate manufacturing elsewhere (Umali and Nikiema, 2002). Caterpillars of Cirina butyrospermi, associated with the trees are a good source of protein for some ethnic tribes such as the Yoruba, Nupe and Tiv who inhabit the central states of Nigeria (Ande, 2004). Although trade in shea tree products has been reported to improve the incomes and living standards of rural farm families and the economies of exporting countries (Popoola and Tee, 2001), its use as a natural wax in storage has not been sufciently reported. Bananas (Musa sp.), like many other fruits and vegetables, are classied as perishable crops. In Nigeria, this crop is abundant and cheap only at certain times of the year and become quite expensive during some parts of the year. The exact gure for estimating post-harvest loses in bananas is difcult to get due to the non-availability of the actual land area under the production of the fruit crop and logistic reasons. Post-harvest losses in banana results from three main sources: mechanical injury, activity of micro-organisms and physiological changes (Liu and Ma, 1985; Lyman, 2000). Because fruits are still living entities after harvest and detachment from the mother sucker, it seems proper to try to slow down the metabolic/ physiological changes that lead to loss of the fruits thus extending the shelf life of such fruits.

Waxing, especially for fruits, plays at least two important roles: (i) it provides repellency for water and other solutions from fruit surfaces and (ii) it reduces the permeability of these solutions through the skin. Water repellency affects the deposition, distribution and retention of chemicals applied to foliage or fruits as solutions or emulsions. Permeability is a major problem when water soluble materials such as calcium need to be introduced into the fruits for desirable effects such as maintenance of rmness. Waxes prevent moisture loss during fruit storage. Although natural waxes on fruits are effective in preventing water loss, the application of commercial wax can further decrease water loss during prolonged storage. A lot of work has been carried out on delaying ripening of bananas and plantains. Some of these have included the use of fungicides and other chemicals to arrest metabolic activity of microorganisms and control diseases (Opadokun and Onwugulu, 1984) and the use of waxes of various compositions (Kolekar, 1988; Marchal, 1990 and Kolattukudy, 2003). The results from these works have been impressive; however, due to non-availability of some of the chemicals and cost, farmers in developing countries may not be able to afford them. The possibility of a residual effect of the chemicals and the safety of their use has necessitated this study to test the effect of locally available and inexpensive coating material (Shea butter oil), that can be used in prolonging the shelf life, ripening and storage of bananas fruits.

Materials and methods

A bunch of banana of Gross Michel cultivar was harvested from a local banana plantation in Minna, Niger State, Nigeria. The bunch was brought into the laboratory of the Department of Crop Production where it was de-ngered. These ngers were washed rst in water to remove latex from the cut ends and other dirt

Potential use of shea nut butter as skin coat for banana particles on the fruits, then washed in a 5% solution of sodium hypochlorite (Parazon - bleach) to disinfect the banana ngers. The ngers were allowed to air-dry for a minimum of eight hours before a few of the ngers were treated with shea butter and then stored. A few uncoated ngers were also stored as control. The butter, derived from nuts of the shea butter tree (V. paradoxom) was purchased from the local Minna market and melted over an open ame. Banana ngers were coated by swapping the ngers with cool melted butter. To prevent the transmission of germs from the hands onto the treated banana ngers, the operator wore rubberized gloves. The banana fingers were first wrapped in paper bags and then placed in polythene bags and stored at 35, 25 and 10 oC. Thermometer probes were inserted in all the storage chambers to monitor storage temperatures. Each treatment was replicated three times. Parameters monitored included days to fruit ripening, fruit colour change as affected by storage temperatures, rate and type of fungus associated with deterioration and palatability (sweetness) of the banana. Statistical Analysis: Data were analyzed using the MINITAB release 14, computer software for Statistics. Analysis of variance (ANOVA) was performed on results for each quality variable to determine the signicant storage method(s) while means were separated using the least significance difference LSD test (Gomez and Gomez, 1983).

75

Deterioration after ripening: Deterioration of the uncoated ngers started after 3 days from when banana ngers were judged to be 100% ripe. Coated ngers lasted an average of 7 days; 4 days more than the uncoated ngers. The deterioration in the treated ngers began rst from the point of attachment to the bunch. This point was observed to have gone yellowish, a sign of ripening much earlier than the pulp of the banana. At the end of 3 days for the untreated and 7 days for the treated ngers, fungal attack was observed. Investigation revealed the presence of strains of Aspergillus avus, A. niger and Rhizoctonia sp. Palatability test: When ripe, bananas were subjected to a palatability test to assess sweetness and texture (softness). A Likert scale of 1-5 was used where 1 represented very poor quality and 5 represented best quality. The result showed that the untreated ngers were rated an average score of 5, while treated ngers were rated an average score of 3, inferring fairness. This was because the placenta of the banana ngers had not softened enough to be noticed at this time. The delay in the number of days to ripining of coated banana fingers was significant (P< 0.05), inferring sufficient delay in ripening compared to untreated ngers. The coating of the ngers with shea butter had caused a reduction in the physicobiochemical processes in the ngers. This result is in agreement with Kolekar et al. (1988) who reported that shelf-life of bananas could be extended by coating them with 0.5-2.0% sucrose ester emulsion. Coating of the bananas ngers impaired rate of respiration as the rubbing of the butter has sealed the lenticels. The results of the taste panel on texture and sweetness is not different from the ndings of Marchal (1990), who reported similar ndings. Esquerra et al. (1993) reported that respiration rate of bananas was high at 35 and 30 oC while it was low at 15 o C. This explains why ngers at 10 oC in the current work did not ripen after the 53 days of storage. Porritt (1974) had opined that the temperature of storage was the one most important external factor that affects the post-harvest shelf life of most perishables. The rate of microbial activity is also enhanced at high temperatures hence the quick deterioration of the banana ngers occurred under high temperature storage. Banana itself is a good base for culturing microbes. This explains the type of fungi found on the fruits. It is however not clear if the fungus observed were present on the banana skin before the coating; introduced from the butter or in the immediate environment or air surrounding the stored fruits. The treatment with sodium hypochloride might have killed only the bacteria and not fungi. The conclusion from this study is that fruit ripening can be delayed when banana ngers are coated with shea butter. The use of this butter could have a good potential for fruit coating. Although low storage temperature environments may not be available for use by farmers in the rural developing nations, retailers in urban
Table 2. Effect of storage environment on taste, aroma and textural quality of banana ngers Storage temperature Taste Aroma Texture 10 oC (Refrigerator) 1.6c* 2.8b 3.6a 1.6c 2.6b 3.6a 1.4c 2.6 3.6a

Results and discussion

Number of days to fruit ripening: Number of days to ripening for both treated and untreated bananas is presented as Table 1. A statistically signicant difference (P< 0.05) was observed between the storage temperatures. Coating treatments and the interaction between storage temperatures and coating was however not signicant. Banana ngers stored in the refrigerator at 10oC did not ripen after 53 days of storage irrespective of the treatment. When removed from this environment though, uncoated banana ngers ripened 5 days later, while the coated ngers remained unripe for 8 days. At 25oC, the coated ngers took 15.7 days to ripen. This was 7 days more than the uncoated ngers that ripened by 8.7 days. Treated banana ngers stored at 35oC took 11.3 days to ripen; this was 6 days more than the untreated ngers stored at the same temperature. Effect of storage temperature on colour of the banana peel: As has been reported ripening was fastest at high temperatures but delayed at low temperature. This also affected the colour of the fruit peel. The development of yellow colour of ngers, indicating ripeness and closely associated with softening of the skin tissue was observed to be earliest at high temperatures. Banana nger stored at 10 oC was green for about 26 days after which ngers started losing chlorophyll and turning brownish green (but still rm). Storage at 10 oC was not too different from that of 25 oC.
Table 1. Effect of wax treatment on number of days to fruit ripening Treatment Coated banana Uncoated banana 10 oC 53 53 Storage temperature 25 oC 15 8 35 oC 11 6

20 C 35 oC

* Likert scale 1-4; where 1 is excellent and 4 is poor.

76

Potential use of shea nut butter as skin coat for banana

Liu, M.S. and P.C. Ma, 1985. Post-harvest problems of vegetables and fruits in the tropic. Asian vegetable Research and Development Centre. 10th Anniversary Monograph series, Shanhua, Taiwan, Republic of China. P14. Lyman, I.A. rd 2000. Processing plantain chips in Honduras. Proceedings of the 3 meeting of IARPB. Abidjan, Cote dIvoire, 27-31 May 1985. Pp162-163 Maranz, S, W. Kpikpi, Z. Weisman, A.D. Sauveur and B. Chapagain, 2004. Nutritional values and indigenous preferences for shea fruits (Vitellaria paradoxa CF Gaertn.) in African Agroforestry parklands. Journal Economic Botany, 58: 588-600 Marchal, I. 1990 Effect of SemperfreshTM, coating on ripening of bananas. Post-harvest News and Information. CAB Abstract Vol. 1, No. 1. Opadokun, J.S. and O.C. Onwuzulu, 1984. Effect of various chemicals on the shelf live of Cavendish bananas. NSPRI Technical Report. No.9. Pp. 83-87. Popoola L. and N.T. Tee, 2001. Potentials of Vitellaria paradoxa Gaertn F. in Agroforestry systems in Benue State. Nigerian Journal Ecology, 16: 20-24. Ugese, F.D., K.P. Baiyeri and B.N. Mbah, 2008. Nutritional composition of shea (Vitellaria paradoxa) fruit pulp across its major distribution zones in Nigeria. Fruits, 63: 163-169 Umali, B.E. and A. Nikiema, 2002. Vitellaria paradoxa C.F. Gaertn. Record from Protabase. Oyen, LP A and Lemmens, RHMJ (Editors) PROTA,Wageningen, the Netherlands.
Received: August, 2011; Revised: November, 2011; Accepted: December, 2011

centers may be able to delay ripening in bananas for more than 50 days when as low as 10o C temperatures are used. The mesophyll part of the shea fruit is edible in most African cultures with the nuts thrown away. A better use of butter from this nuts bring added value to shea butter. As this butter is often used as medicines in developing economies and skin treatments in developed economies, this infers that its use cannot be harmful to health. Its use as a coating wax can be an additional benet for developing economies as that of Nigeria.

References
Ande, A.T. 2004. The pupal habits of Cirina fordea, a leading food insect in Kwara State Nigeria. J. Sustainable Trop. Agric. Res., 9: 97-100. Esguerra, E.B., K. Kawada and H. Kitagawa, 1993. Responses of Senorita and Cavandish bananas to postharvest control treatments. ASEAN Food J., 8(2): 6-9. Gomez, S.S. and A.A. Gomez, 1983. Statistical Procedure for Agricultural Research. IRRI Philippines, 2nd ed., pp. 680 Kolattukudy, P.E. 2003. Natural waxes on fruits. <http://postharvest. tfrec.wsu.edu/REP2003A.pdf> Kolekar, T.G. 1988. Shelf-life extension of banana by use of sucrose ester formulation. Indian Journal Plant Physiology, 31(1): 16-20.

Journal

Journal of Applied Horticulture, 14(1): 77-80, 2012

Appl

Allelopathic effect of orchard soils on seedling growth of rough lemon (Citrus jambhiri Lush.)
R.P.S. Dalal1*, Navjot, A. Thakur, A.S. Sidhu and J.S. Brar Punjab Agricultural University, Regional Station, Bathinda 151001, India. 1 Present address: Department of Horticulture, CCS HAU, Hisar (Haryana), India. *E-mail: dalal08@rediffmail.com

Abstract
An experiment was conducted to study the allelopathic potential of old orchard soils on the seedling growth of rough lemon. Soils from the root zone spheres of eight orchards of mango, aonla, peach, pomegranate, citrus, pear, ber, guava and virgin soil as a control was used for raising the seedlings. The rough lemon Jatti khatti seedlings of one and a half year old raised in aonla, ber and peach orchard soil as growing media showed the reduction in shoot length (40-50%), leaf number (46-63%), leaf area/ plant (62-69%) and shoot dry weight (79-83%). The root length was most inhibited by ber, aonla and peach orchard soils. The percent reduction in root dry weight (11.23- 34.48%) of the seedling was not in equal proportion to reduction in root volume (42.55- 55.86%). Root dry weight density varied between 0.55-0.96 g mL-1 and root: shoot ratio between 1.42-1.82. Whereas, in citrus, mango, pomegranate, ber and guava orchard soils, the percent reduction in root dry weight was in equal proportion to root volume and root dry weight density varied between 0.41-0.49 g mL-1 and root: shoot ratio between 0.44-0.72. The shoot and root growth of the seedlings was at par when raised in citrus and pomegranate orchard soil as growing media. Leaf N and P contents increased, whereas, Ca and Mg decreased in all the orchard soils except citrus and pomegranate orchard soils as growing media. Leaf Fe, Cu and Mn contents in all the orchard soils as growing media were in toxic range except citrus and pomegranate orchard soils. Overall, the orchard soils of deciduous fruit plants showed more allelopathic effect than the soils growing evergreen fruit plants in citrus cultivation. Key words: Allelopathy, Citrus jambhiri, old orchard soils, seedling growth, nutrients.

Introduction
Allelopathy is an important mechanism of plant interference and is mediated through the addition of chemicals to the plant environment. However, the difculty in replanting fruit trees following the removal of old orchard has been recognized for many years in U.S.A. and Europe. Most of the research indicated that numerous allelopathic compounds are released into the environment by root exudations, leaching, decomposition and volatilization (Rice, 1984). In monoculture of annuals and cultivation of perennial plants in orchards and grasslands where the same substrate is used many times, there is more build up of homogenous metabolites and plant residues in the soil. Besides, living plants exuding the allelochemicals, microbial decay of plant residue also release the toxic metabolites into the soil (Politycka, 2005). They are present in the roots, rhizomes, stems, leaves, owers, pollens, fruits and seeds but the leaves are the major source. Their concentration varies with age, season, plant parts and growth habit etc. (Narwal, 1994). The phytotoxic potential of these chemicals depends on plant type, activity of microora, their release and removal from the soil solution or immobilized by the plant uptake, adsorption to the soil particles and degradation by micro-organism (Kobayashi, 2004). Allelochemicals impair the physiological function of the cell directly or indirectly, thereby retard plant growth and cause soil sickness in various fruit crops (Hassan et al., 1989 and Arora et al., 2002 in citrus; Ercisli and Turkkal, 2005 in walnut; John et al., 2007 in pomegranate; Sharma et al., 2000 in mango; Oudhia, 2001 in guava; Alshahrani et al., 2009 and Arya et al., 2011 in ber

and aonla). However, most of the research has been conducted on fruit trees-eld crops and weedsfruit trees interactions, studies on allelopathy aspects of interaction among fruit tree species is still lacking. Understanding of interaction will help in solving the problem in success of replanting. Keeping in view the above facts, the present investigation was carried out to nd out the allelopathic effect of different old orchard soils on the seedling growth of rough lemon syn. Jatti khatti Lush.

Materials and methods

The experiment was carried out at PAU, Regional Station, Bathinda (Punjab) during 2008 and 2009. Bulk soils were collected from virgin soil and root zone sphere (0-40 cm depth and 30-60 cm radial distances from the trunk) of three plants, each aged 15-19 years from mango, aonla, peach, pomegranate, ber, pear and guava orchard during 2nd fortnight of February. The soil collected from each treatment was mixed thoroughly and analyzed for pH, EC, OC, phosphorus and potassium content. pH ranged from 8.00-8.21; EC - 0.15-0.23 dsm-1; OC- 0.320.45%, P - 11-17 kg ha-1 and K- 161-256 kg ha-1. The roots, which were collected from root zone sphere site, were chopped, dried and these chopped root bits were mixed with the soil @ 60g/pot. The cemented pots of 40 cm diameter and 40 cm depth capable of holding 25 kg air dried soil were lled with soil and saturated with the water before sowing of the seeds. The freshly extracted seeds of rough lemon (Citrus jambhiri Lush.) collected from single tree were sown during 1st week of March and the pots were placed in open nursery area. Three pots were maintained per treatment and after germination, 30 seedlings

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Allelopathic effect of orchard soils on seedling growth of rough lemon area/ plant (62-69%) and shoot dry weight/ plant (79 - 83%). Rough lemon seedlings behaved differently in different orchard soils. This may be due to differential response of rough lemon rootstock to different old orchard soils as it is very sensitive to allelo-chemicals (Singh and Achhireddy, 1987) and secondly, the quantitative as well as qualitative differences in the accumulation of allelochemicals by different fruit plants and their phytotoxic potential. These results are in conformity with those of Narwal (1994) that concentration of allelochemicals varies with season, plant parts and growth habit and phytotoxic potential of these chemicals depends upon plant type, their release and removal from the soil particles (Kobayashi, 2004). Seedlings raised in citrus and pomegranate orchard soil behaved similarly in all the growth aspects as there was same proportion of decrease in shoot and root growth and biomass production as compared to control. Saroj et al. (2002) reported that ber leaf extract had variable response on mustard, cluster bean and wheat vigour index. Jadhav (2003) found that leaf extract of aonla inhibited the growth of the eld crop. Arya et al. (2011) reported the inhibited growth of mustard, moth bean, cluster bean and brinjal with the leaf extract of ber and aonla. Similarly, Alshahrani et al. (2009) reported that leaf extract of Zizyphus Spina- Christi was inhibitory to the growth of Prosopsis juliora. Root growth: Root growth of the seedlings was found signicantly altered when raised on various old orchard soils (Table 2). The minimum primary root length was measured in seedlings raised in peach orchard soil which was statistically at par with ber, aonla and pear orchard soil. The maximum root length of seedlings was measured in control which was at par with citrus, mango, pomegranate and guava orchard soil. Root volume was least in ber orchard soil followed by peach orchard soil whereas it was highest in control. Similar results were earlier reported by Arora et al. (2009) that maximum primary root density was observed in mango and guava and minimum in ber orchard soils irrespective of citrus rootstocks. Root dry weight of the seedlings raised in virgin soil (control) was at par with peach orchard soil. The least root dry weight was measured in ber orchard soil which was at par with guava orchard soil. The seedlings raised in citrus and pomegranate orchard soil attained almost similar root dry weight 4.07 and 4.13g/ plant, respectively and in mango and aonla orchard soil 4.93 & 4.87g/ plant, respectively. Whereas, in peach, aonla and pear orchard soil, the root dry weight decreased from 11.23-34.48% and root volume from 42.55-55.86%. Hence root dry weight density varies from 0.55-0.96g/mL and root: shoot

were maintained/ pot. The seedling irrigated as and when required with equal amount of canal water to prevent leaching from the bottom of the pots. Weeds were removed manually and insects were controlled chemically. These seedlings were allowed to grow for about one and half year. The seedlings were uprooted, washed and separated into shoots and roots. The length of shoots and roots were measured with meter scale and shoot diameter at 2-inch height with Vernier Caliper. Root volume was determined with water displacement method and leaf area was measured with leaf area meter. Dry weight was observed after drying in an oven at 60 0C till the constant weight was noticed. Root dry weight density was calculated by the ratio of root dry weight to root volume and expressed as g/mL. Leaf samples were collected during September, washed, oven dried, ground and digested with diacid (H2SO4: HClO4) in the ratio of 4:1 for macronutrients contents, and with (HNO3: HClO4) for micronutrients. Nitrogen was estimated with Nesselers reagent method and phosphorus by Vando Molybdo Yellow colour method, potassium with ame photometer and Ca and Mg colorimetricaly. Micronutrients were estimated with Atomic Absorption Spectrophotometer. Trials were carried out in a completely randomized design with three replications. Data were subjected to ANOVA and means were compared by Duncans multiple range test.

Results and discussion

Shoot growth: The shoot growth of the rough lemon rootstock seedlings in terms of shoot length, stem diameter, leaf area, shoot dry weight and total dry weight was signicantly inhibited by old orchard soils (Table 1). The maximum reduction in shoot length, stem diameter, number of leaves was observed in seedlings raised in aonla orchard soil followed by ber orchard soils. Whereas, the maximum reduction in leaf area and total dry weight of the seedlings was found in ber orchard soil followed by aonla grown orchard soil. The minimum shoot dry weight of seedlings was observed in peach orchard soil which was at par with aonla and ber grown soil. Shoot dry weight and total dry weight of the seedlings raised in mango, pear and guava orchard soils was found at par with each other. The citrus orchard soil also exhibited signicantly inhibitory effect on the growth of rough lemon seedling i.e., shoot length, stem diameter, leaf count, leaf area, shoot dry weight and total weight which were at par with pomegranate orchard soil. Rough lemon seedlings raised in aonla, ber and peach orchard soil recorded the maximum reduction in shoot length (40-50%), leaf number (46-63%) and leaf

Table 1. Effect of different old orchard soils on the shoot growth and dry weight of Rough lemon rootstock seedlings Leaf area/ Shoot dry wt./ Total dry wt. Treatments Shoot length Stem diam. Leaf count/ Leaf area/ leaf (cm2) plant (g) (g) (Growing media) (cm) (cm) plant (Nos.) plant (cm2) Virgin soil (control) 68.60f 0.84e 106.67e 877.21f 8.22e 18.99d 25.38f Citrus orchard soil 55.35e 0.65d 85.67d 664.15e 7.75d 9.21c 13.27e ab cd d d b b Mango orchard soil 37.45 0.59 87.00 533.25 6.13 6.77 11.70de a a a ab c a Aonla orchard soil 34.01 0.52 39.50 287.51 7.28 3.42 8.29ab c a bc bc ab a Peach orchard soil 41.76 0.54 57.33 330.80 5.77 3.13 8.82bc d bc c c a b Pear orchard soil 44.97 0.58 66.00 376.98 5.71 6.88 11.09cde ab a ab a a a Ber orchard soil 37.72 0.52 48.50 267.56 5.52 3.95 6.47a e d d e cd c Pomegranate orchard soil 56.81 0.66 84.67 627.68 7.14 8.89 13.03e d cd d d b b Guava orchard soil 46.92 0.62 92.33 564.69 6.11 6.47 9.82bcd Mean (n=12) separated by Duncans Multiple Range Test. Values followed by different letter within a column are signicantly different (P<0.05) from other values in the same column.

Allelopathic effect of orchard soils on seedling growth of rough lemon ratio between 1.42-1.82 which may be either due to change in root morphology/ architecture and accumulation of allelochemicals and mobilization of more metabolites from shoot to roots under stress caused by allelochemicals produced by these plants or both. Hence, it seems that these plants produced chemicals which accumulates in roots and causes stress to seedlings hence increased weight proportionally. A shift in relative allocation of biomass to roots has been earlier observed under condition of environmental stress (Alshahrani et al., 2009; Rutherford and Powrie, 1993). In citrus, mango, pomegranate and guava, the percent reduction in root dry weight was in equal proportion to root volume; hence root dry weight density varied between 0.41 to 0.49g/ mL. This may be due to less difference in partitioning of metabolites in seedling raised in these orchard soils. Hassan et al. (1989) reported that allelopathy is at least partly involved in the citrus replant problem. John et al. (2007) reported that the nature and degree of allelopathic effects of trees varied with crop species. Aonla, peach, pear are the winter deciduous and shed their leaves in winter months and due to slow decomposition under low temperature in these months it may lead to accumulation of toxins in the soil. Ber is the summer deciduous which sheds its leaves in month of May followed by heavy monsoon rain in July, so there in very fast decomposition of leaves due to high temperature and rainfall and hence more accumulation of toxins in the soils. Earlier, Narwal (1994) reported that leaves are the major source of allelochemicals and concentration varies with season and growth habit. Citrus, mango, pomegranate and guava plants are evergreen and there is very less litter fall and hence there is less production of toxins which may either be degraded or leached out with the passage of time so there is less inhibitory effect by these plants.

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The accumulation of allelochemicals in soil to the toxic level is decided with difference between the speed of allelochemicals release and their degradation (Wojcik Wojtkowiak et al.,1998). These results are in conformity with those of Arya et al. (2011), Oudhia (2001) and Sharma et al. (2000) in eld crops with mango, guava, ber and aonla leaf leachates. Leaf macronutrients: Leaf macronutrients contents i.e., N, P, K, Ca and Mg differed signicantly among the various treatments (Table 3). The maximum N content in the seedlings of rough lemon was estimated when grown in guava orchard soil which was at par with mango and pear orchard soil and it was minimum in citrus and pomegranate orchard soil, which was at par with control. Singh and Achhireddy (1987) found that rough lemon grown with Lantana had higher N content than when grown alone. Seeding raised in pomegranate orchard soil contained P which was at par with control and citrus orchard soil. Maximum P accumulation was in the seedling of rough lemon raised in ber and peach orchard soil followed by guava (0.20%) and aonla orchard soil. Potassium content varied between 1.75 to 1.92 %. The maximum value was observed in ber and minimum in guava orchard soil. Buchholtz (1971) reported reduced N, P and K uptake by corn due to allelopathic effects of quack grass. He further stated that the inhibition or stimulation of N, P and K uptake in both corn (Zea mays) and soybeans (Glycine max L.) by several weed residue was not consistent, and relationship between inhibitory effects and nutrients uptake were not found. Calcium content was found maximum in citrus orchard soil raised seedling closely followed by control. The minimum leaf Ca content was observed in ber orchard soil, whereas in pear, peach, aonla and guava orchard soil the rough lemon seedlings leaf Ca content

Table 2. Effect of different old orchard soils on the root growth, root dry weight and root: shoot ratio of Rough lemon rootstock seedlings Treatments Primary root length Root volume Root dry wt. Root dry wt. density Root: Shoot (Growing media) (cm) (mL) (g) (g/ mL) dry wt. Ratio 13.16f 6.41e 0.487a 0.337a Virgin soil (control) 39.61c c d bc a Citrus orchard soil 39.41 8.44 4.07 0.482 0.442b c e cd a Mango orchard soil 38.37 10.52 4.93 0.469 0.728d c bc cd c Aonla orchard soil 39.61 6.42 4.87 0.758 1.424e a b de d Peach orchard soil 32.52 5.94 5.69 0.958 1.818f ab cd bc ab Pear orchard soil 34.76 7.56 4.20 0.555 0.610c a a a b Ber orchard soil 33.64 4.28 2.51 0.586 0.635cd bc e bc a Pomegranate orchard soil 37.17 10.57 4.13 0.400 0.464b bc c ab a Guava orchard soil 37.82 6.82 3.34 0.490 0.516bc Mean (n=12) separated by Duncans Multiple Range Test. Values followed by different letter within a column are signicantly different (P<0.05) from other values in the same column Table 3. Effect of different old orchard soils on the leaf nutritional status of Rough lemon rootstock seedlings Treatments (Growing media) Virgin soil (control) Citrus orchard soil Mango orchard soil Aonla orchard soil Peach orchard soil Pear orchard soil Ber orchard soil Pomegranate orchard soil Guava orchard soil C D (5%) N 1.66 1.47 2.33 2.14 1.90 2.24 2.08 1.47 2.39 0.21 P 0.12 0.12 0.14 0.18 0.22 0.16 0.22 0.10 0.20 0.06 Macronutrients (%) K 1.82 1.77 1.72 1.88 1.77 1.77 1.92 1.82 1.75 NS Ca 3.54 3.62 3.22 3.15 3.10 3.10 2.80 3.45 3.27 0.14 Mg 0.70 0.60 0.58 0.54 0.54 0.48 0.38 0.62 0.52 0.11 Zn 15 15 16 14 14 13 13 18 12 1.4 Micronutrients (ppm) Fe Cu 62 98 65 93 71 104 79 134 75 174 81 142 109 124 62 90 62 104 12 18 Mn 16 19 15 21 27 21 32 19 20 3.0

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Allelopathic effect of orchard soils on seedling growth of rough lemon

ranged from 3.10-3.27%. The maximum Mg content was found in control which was statistically at par with citrus and pomegranate orchard soil raised seedling. The minimum leaf Mg content was observed in ber orchard soil. The leaf Mg content ranged from 0.48- 0.54% in pear, aonla and peach orchard soil. Ca and Mg uptake was inhibited by all the treatments except citrus and pomegranate orchard soils and the inhibition was more where there was more growth inhibition. It was observed that all orchard soils as growing media played an important role in decreasing the capacity for cation uptake except citrus and pomegranate and has a positive effect on uptake of plant nutrients such as N, P, Fe, Cu and Mn. Similar results were earlier reported by Ercisli and Turkkal (2005). Leaf micronutrients: The maximum leaf Zn content in the seedlings of rough lemon was observed in pomegranate orchard soil which was signicantly better than the other treatments and minimum was recorded in guava orchard soil. Maximum leaf Fe content was in seedlings grown in ber orchard soil, whereas in other treatments it ranged from 62-79 ppm only. The maximum Cu content was found in seedlings raised in peach and minimum in pomegranate orchard soils. In citrus, mango and guava orchard soil, the leaf Cu content varied from 93-104 ppm, whereas, it varied from 124-142 ppm in ber, aonla and pear orchard soil raised seedlings. Manganese content of leaves was found maximum in ber orchard soils which was at par with peach orchard soil and was minimum in mango orchard soil grown seedlings. Overall, Fe, Cu, and Mn contents were higher than reported in literature which may have caused toxicity to the rough lemon seedlings and hence reduced the growth. Thus, our study shows that where growth was inhibited more there was more nutritional imbalance such as in ber, peach, aonla, pear, guava and mango orchard soils. The decrease in nutritional status in citrus and pomegranate orchard soils was less as compared to other growing media. This may be either due to dilution factor or high uptake. Thus, it is reasonable to assume both direct and indirect effects of allelochemicals on plant water relationship and accumulation of stress producing chemicals, which in turn cause reduction in plant growth and translocation of metabolites and imbalance/ inhibition/ stimulation of nutrients. It may be concluded that citrus rootstock tested showed different and relatively consistent pattern of sensitivity to old orchard soil. So, it can be inferred that allelochemicals may be selective in their action, or plants may be selective in their response. Allelochemicals produced by the different fruit plants affected the citrus rootstock growth in one or the other way by inhibiting root/ shoot growth or both. This study further indicates that deciduous fruit plants like ber, peach, aonla and pear have more allelopathic potential than evergreen fruit plants viz., citrus, mango and guava in citrus cultivation. This study may help to know the fruit tree to fruit tree interaction and to resolve the problem faced in reorcharding.

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Received: May, 2011; Revised: June, 2011; Accepted: August, 2011